manual tgge mini 6 - cultek · the tgge system allows strictly defined linear temperature gradients...

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TGGE Manual Ver 6.0 16.01.02

TGGE System(order number 024-000)

ManualVersion 6.0

January 2002

Please read this manual carefully beforeusing the instrument

Biometrabiomedizinische Analytik GmbH

Rudolf-Wissell-Str. 30, D-37079 GoettingenTel.: 0551/50 686-0; Fax: 0551/50 686-66

email: [email protected]

http://www.biometra.com

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1. What’s new in this manual

The current version of the TGGE manual reflects the strong progress that has been made withthe TGGE method. New insights concerning handling and separation conditions have beenintegrated in the proven step by step protocol.

The major news are:

1) The volume of thermal coupling solution is a critical factor for an even migration frontand should be kept as small as possible (section 5.4)

2) Overlaying the gel with buffer should be omitted (section 5.4.2)

3) The gel cover film should be applied right after putting the gel on the block (section 5.4.2)

4) Identical salt concentration in all samples is important for an even migration front.

5) Extended Silver staining protocol for optimum gels (chapter 9)

6) Electrophoresis conditions for mutation analysis and microbial diversity analysis (section5)

7) Precast gels for TGGE (section 4.1)

8) Gel casting stand for casting 5 gels in parallel (024-028)

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2. Safety instructions

• Do not fill buffer chambers above marking for maximum level

• If buffer has been spilled on the electrophoresis unit, clean it carefully before start ofelectrophoresis

• Never run device without gel cover plate

• The thermoblock is covered with a Teflon film. Avoid damaging this film.

• For cleaning of the thermoblock do not use aggressive chemicals or strong detergents.

• Do not use paraffin oil or kerosene on the thermoblock.

• In case of strong condensation under the safety lid stop run, dry instrument and re-start

• Switch off power before removing the safety lid

• Do not move instrument during operation

• Do not lift the electrophoresis unit by holding it on the white frame. Instead, lift thelower part of the device (red corpus).

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3. Introduction

Temperature Gradient Gel Electrophoresis is a powerful technique for the separation ofnucleic acids or proteins. The TGGE method, which is covered by patents, uses thetemperature dependent changes of conformation for separating molecules (for review seeReference 1).

Since the introduction of the first commercial available TGGE apparatus in 1989, temperaturegradient gel electrophoresis has gained high interest in scientific and clinical researchlaboratories due to the unprecedented resolution capability and easiness of analysis. The rangeof scientific publications using the TGGE method is broad and covers all disciplines whichuse molecular biology methods: e.g. Oncology2-4, Virology5,6, Immunology7,8, RNA ViroidResearch9-12, Prion Research13, Population Analysis14-15. The TGGE method has also beenused for quantitative analysis in industry16-17 and for conformational analysis of proteins18-19.

3.1 Principle of the method

Conventional protein or nucleic acid electrophoresis separates molecules according to theirsize or charge. TGGE adds a new parameter for separation, namely the melting behavior ofa molecule. The melting behavior is determined by primary sequence and secondary andtertiary structure of the molecule and can be changed by external influences like temperature,salt concentration, pH etc.

During electrophoresis the sample migrates along a temperature gradient. As the temperaturerises the molecules start to denature. Working with PCR fragments for exampleelectrophoresis starts with double stranded molecules. At a certain temperature the DNAstarts to melt, resulting in a fork-like structure (partial single strand, see Figure 1). In thisconformation the migration is slowed down compared to a completely double stranded DNAfragment (of same size). Since the melting temperature strongly depends on the basesequence, DNA fragments of same size but different sequence can be separated. This is usedin mutation detection where PCR fragments of identical size but different sequence areseparated. Thus TGGE not only separates molecules but gives additional information aboutmelting behavior and stability.

cold

warm

elec

trop

hore

sis ds DNA

partial ss

ss DNA

Figure 1: Different conformations of DNA during temperature gradient gel electrophoresis.

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3.2 Special features of the Biometra TGGE System

The most powerful characteristic of the Biometra TGGE system is the high reproducibility ofthe temperature gradient. In contrast to conventional systems using chemical gradients(DGGE) the temperature gradient of the Biometra TGGE system establishes the samedenaturing gradient time after time after time. The microprocessor driven gradient block ofthe TGGE System allows strictly defined linear temperature gradients with high resolutionand reproducibility.

Because of the small amount of material used for separation, DNA or RNA fragments appearas fine bands which can be clearly distinguished from each other. Even complex band patternscan be analyzed due to the high resolution capability of the gradient block. Comparing theTGGE method with other screening methods like SSCP the superior performance of theTGGE method becomes evident20-22.

The Biometra TGGE system is available in two formats. The standard TGGE “mini” system(024-000) operates small gels and is therefore ideally suited for fast, serial experiments. TheTGGE maxi system (024-200) provides a large separation distance and allows high parallelsample throughput.

With the Biometra TGGE system it is very easy to separate samples either parallel orperpendicular to a temperature gradient. All that has to be changed is the position of thebuffer tanks. Whereas perpendicular TGGE is mainly used for the optimization of separationconditions, parallel TGGE allows fast analysis of multiple samples.

PERPENDICULARTGGE

Temperature gradient is perpendicular to the electrophoreticmigration:

One sample is separated over a broad temperature range todetermine the optimum temperature gradient or to analyzetemperature dependent changes in conformation

PARALLEL TGGE Temperature gradient is parallel to electrophoretic migration:

multiple samples are separated in parallel

Figure 2: Typical results after perpendicular TGGE (A: temperature gradient from left toright) and parallel TGGE (B: temperature gradient from top to bottom).

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3.3 Before you start: The TGGE flow chart of success

TGGE is a powerful technique to separate molecules of same size, but different sequence.Nevertheless, every DNA fragment has its own characteristics and three steps have to betaken before successful analysis of multiple samples in parallel TGGE can begin. Each of thefollowing steps is described in detail in section 8.

Step 1 Check the sequence of your PCR fragment in Poland analysis. The Poland computerprogram (http://www.biophys.uni-duesseldorf.de/POLAND/poland.html) calculates themelting behavior of dsDNA molecules. Poland analysis can predict, whether a fragment issuited for TGGE or not. Analysis is available online and free of charge.

Melting profile is okPoland analysis shows asatisfying profile.

Proceed with step 2

Melting profile is not okIf Poland analysis shows that the fragment in its currentstate is not suited for TGGE, optimize your primer design.Never try to separate samples in TGGE if the calculatedmelting profile is not ok. Back to step 1

Step 2 If the Poland analysis shows a suitable melting profile you should test separationconditions in a perpendicular TGGE. In perpendicular TGGE, a large aliquot of the sampleruns over a broad temperature range. The result of parallel TGGE allows identification of thetemperature gradient for parallel analysis.

Perpendicular gel isokPerpendicular TGGEshows a nice meltingcurve.

Proceed with step 3

Perpendicular gel is not okIf perpendicular analysis does not show the expectedmelting profile, check sequence again in Poland analysis.Also check purity of chemicals and electrophoreticconditions.Do not try samples is parallel TGGE, if the perpendiculargel does not show a defined melting curve.Back to step 2

Step 3 Set up a parallel gel with the temperature gradient derived from the perpendiculargel. Separation can be optimized by varying the temperature gradient and voltage.

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3.4 System Overview

Safety lid

Buffer chambers

Electrophoresis unit

controller cord plug

Figure 3: TGGE Electrophoresis unit

The TGGE System contains all components necessary to get started. All kinds of TGGEapplications (parallel or perpendicular TGGE, Constant Temperature GE, Time resolvedTGGE) can be run with the Biometra TGGE System. A broad range of different glass platesare available and can be ordered from Biometra or your local distributor (see 6.3. OrderInformation).

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3.5 Components of the TGGE System

TGGE Controller with integrated power supply, 100 program storesand control function of temperature and electrophoresis conditions

024-001

TGGE-Electrophoresis unit with 2 removable buffer chambers,Peltier-element powered gradient block and control cable

024-002

TGGE Starter Kit contains 024-003

3 plane glass plates 024-021

1 glass plate with 8 slots for parallel TGGE 024-022

1 glass plate with 1 rectangular slot for

perpendicular TGGE

024-023

1 glass pate with 12 slots for parallel TGGE 024-025

Electrophoresis wicks (100/pkg) 024-015

Polybond film (25/pkg) 024-030

1 Cover glass plate and 10 cover films 024-031

1 Acryl-Glide (sample)

Manual

When unpacking your System please check if all parts are included. If parts are missing callBiometra or your local distributor.

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4. Preparing TGGE gels

The TGGE system (024-000) contains all components needed for casting custom gels (glassplates, clamps etc.). Alternatively, special precast gels for TGGE are available.

4.1 Precast gels for TGGE

Precast gels for TGGE with 13 slots for 5µl each are available (order number 024-040). Thegels are completely dehydrated allowing long term storage. The gel matrix has aconcentration of 8% Polyacrylamide. Prior to use the gels are rehydrated in the desired buffer.

4.1.1 Technical specification

Ordernumber

Quantity Dimensions Slots Polyacrylamide Storage

024-040 10 8,3 x 8,8cm 13 (5µl) 8% -20°C > 1 year

4.1.2 Content

Included Additionally required

Clean gels

Paper sheets

Manual

Equilibration buffer (1 x TAE, 8M urea, 2% glyerol)

Incubation tray

Rocking platform

4.1.3 Rehydration of precast TGGE gels

1) Fill tray with equilibration buffer. The final buffer composition in the gel will be identicalto the equilibration buffer. Use at least 20ml.

2) Place clean gel upside down on the buffer surface. With gentle agitation (rockingplatform) the gels floats freely on the buffer ensuring efficient equilibration. The backsideof the gel should not get in contact with equilibration buffer. Make sure that the gel doesnot adhere to the bottom of the tray.

3) Equilibrate gel for 90 minutes.

4) Remove gel from the buffer tray and put it on a flat surface (upside up). Remove residualbuffer carefully from the gel surface by sliding along with the edge of a paper sheet(included). It is very important that the gel surface is perfectly dry (sliding along with thepaper, you should hear a squeaking noise).

4.1.4 Applying precast gels on the TGGE thermoblock

The following steps are not different from using handcast gels. However, the following pointsshould be taken into consideration.

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• Make sure that the backside of the gel is absolutely clean.

• Use not more than 150µl thermal coupling solution (0,01%Triton X100).

4.2 Handcast gels for TGGE

The thinness of the polyacrylamide gel makes it necessary to cast the gel on a gel support film(Polybond film, Order Number: 024-030). The gel will stay on this film throughout the wholeprocedure (including silver stain).

4.2.1 Components of gel cuvette

Each sandwich for casting TGGE gels consists of three components:

1 Cover glass plate without spacers (bonding plate)

2 Polybond film

3 Glass plate with slot formers and spacers (different types of slots available).

Glass plate with slot fomer and spacers

(024-022)

Bonding plate(024-021)

Poybond support film(024-030)

Figure 4: TGGE glass plates

4.2.2 Treatment of glass plates

• Glass plates must be dry and free of any dirt or dust. Please wear powder-free gloves duringcleaning and assembly of gel sandwich, to prevent contamination (which will interfere withsilver staining).

• Do not use strong acidic or basic solutions or organic solvents for cleaning the glass plates.

• Do not apply mechanical stress on slot formers (i.e. brushing or rubbing)

• Do not incubate glass plates over night in cleaning solutions!

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Pretreatment of glass plate with spacer and slot former (not bonding plate!)

• The glass plate with spacer and slot former must be carefully treated with an repelling agent(for example Acryl-Glide, sample included). This is essential for easy removal of thepolymerized gel from the glass plate with slot formers.

• Drop about 0.5 ml of solution onto the plate and spread it carefully especially between the slotformer. Wait 5 min, than polish the plate with soft tissue to remove residual solution!

• Important: Do not drop repelling solution on the spacers of the glass plate! This will lead toleakage during polymerization.

• Treatment of glass plates (not bonding plate!) with repelling solution should be repeated priorto each gel casting.

• Clean spacers with ethanol before assembling the glass plate sandwich.

• Apply some silicone grease onto the spacers (to prevent leakage).

Note: Treatment of the glass plate with slot formers should be repeated prior to every use.

Note: Never treat spacer with repelling agent because this will cause leakage. Don not treat thebonding plate with repelling agent.

4.2.3 Polybond film

• Use original pre-cut Polybond support film which perfectly fits onto the gradient block.

• The Polybond support film has a hydrophilic coating. The gel matrix binds covalently to thesupport film. (note: In earlier batches, Polybond film was coated only on one side. Today,either side can be used for gel casting. The orientation of the film is not important).

• Remove protecting paper sheet from polybond film prior to assembly of sandwich. HandlePolybond film only with powder-free gloves .

• Attach Polybond film firmly to the bonding glass plate by gently rubbing with a soft tissue.To improve adherence to the bonding glass plate, you may put a drop of water on the plate.Since the Polybond film is hydrophilic, it will readily stick to the wet glass plate.

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4.3 Assembling the glass plate sandwich

• Assemble the Acryl-Glide treated glass plate with spacer, the Polybond film and theBonding glass plate as shown.

Glass plate with slot fomer and spacers

(024-022)

Bonding plate(024-021)

Poybond support film(024-030)

Figure 5: Set up of gel sandwich for TGGE:1) Attach Polybond film to bonding glass plate.

2) Put glass plate with slot formers on the polybond film.3) Fix gel sandwich with three clamps (or use gel casting stand 024-028).

Clamp

ClampClamp

Figure 6: Final setup of the gel sandwich Figure 7: Gel casting stand

Note: For convenient casting of 5 gels in parallel we recommend gel casting stand 024-028,(Figure 7).

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4.4 Preparing gel solution

TGGE gels contain a high concentration of urea and therefore must not be stored at lowtemperatures (urea will precipitate).

Polyacrylamide is a toxic substance. Skin contact or inhalation must bestrictly avoided. Wearing laboratory gloves is mandatory during all steps.Please carefully read the safety sheets of the chemicals prior to use andbehave accordingly.

Each gel sandwich contains approx. 2.5 ml polyacrylamide solution. We recommend toprepare 10 ml solution to pour 3-4 gels at a time. Gels should be used within 24 hours afterpolymerization. To prevent gel drying, wrap polymerized gels into saran foil or wet plasticbags. Wet paper towels may be used for short time storage.

The below gel composition is intended for mutation analysis with the TGGE test Kit (024-050). Gel protocols for other applications can be found in section 5.

Protocol for 10 ml gel solution (3 gels)

Volume /amount

Finalconcentration

Urea 4,8 g 8M

40% Acrylamide/Bis-Acrylamide (37,5 : 1) 2 ml 8%

10 x TAE 1 ml 1 x

40% Glycerol 500 µl 2 %

Adjust with aqua bidest to 10 ml

• stir solution at 50°C until urea is completely dissolved

• Carefully degas gel solution

• Let solution cool down to room temperature and start polymerization with

APS (4%) 40 µl

TEMED 9 µl

• Once TEMED and APS have been added, the following steps must be performed withoutdelay.

• Load gel solution in a syringe (avoid bubbles)

• Attach 0,45µm sterile filter

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Note: Pouring gels from the same batch solution will increase reproducibility. Afterdissolution of urea batch solution may be stored up to 2 weeks at room temperature.

4.5 Pouring gels

Figure 8: Pouring polyacrylamide gel.

• Hold gel sandwich at an angle of 45° when pouring. The solution should run slowly alongone side of the plate sandwich to avoid air bubbles. Turn gel sandwich into verticalposition.

• Polymerization of gel for 1 - 1.5 h or over night at room temperature. The sandwich shouldstand up vertically and must not be moved during polymerization.

• Use gels within 24 hours.

Note: Gels must not be stored at low temperatures (i.e. refrigerator) because this will lead toprecipitation of urea.

4.6 Disassembling the gel sandwich

• Remove the clamps from the plate sandwich.

• Remove the bonding glass plate from the sandwich by carefully sliding it sidewards. Thegel polymerized to the Polybond film should adhere to the glass plate with slot formers.

• Slowly take off the Polybond film with the adhering polyacrylamide gel from the otherglass plate. In the area of the slot former remove the Polybond film very carefully toavoid any damage to the slots.

• Do only use perfect shaped slots because otherwise the bands will be distorted.

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5. Electrophoresis with the TGGE System

Since in TGGE molecules are separated according to their primary sequence, there are manypotential fields of application. For some applications TGGE has become already the methodof choice. Currently, there are two major fields: Mutation analysis in PCR fragments anddiversity analysis of complex bacterial samples. The main difference between these twoapplications is that in the former case only one or two PCR fragments (mutant and wildtype)are separated per lane, whereas in the latter case a mixture of many (up to 150) fragments areseparated in one lane. While sample throughput is always an important aspect, long separationdistance is mainly necessary, if many bands are to be separated. Therefore the TGGE maxisystem (024-200) is the system of choice for diversity analysis, whereas the standard system(024-000) allows short electrophoresis times and provides high serial sample throughput formutation analysis.

5.1 General remarks

In contrast to conventional electrophoresis techniques like PAGE or agarose gelelectrophoresis the migration behaviour of samples in TGGE depends very strongly on thetemperature gradient. Better separation in TGGE is NOT achieved by longerelectrophoresis times! Once the samples start to melt their migration is almost stopped.Therefore extended migration times will rather lead to fuzzy bands than to a better resolution.To improve resolution (i.e. distance between two bands in one lane) the temperature gradienthas to bee adjusted (see section 8.5).

In general migration of samples in TGGE depend on

• Temperature gradient

• Buffer system

• Gel composition

• Voltage

• The kind of sample, e.g. protein, nucleic acid, fragment size

• The kind of application, e.g. parallel or perpendicular TGGE

• Sample preparation, e.g. high salt or low salt preparation

Important: The TGGE controller is designed to control voltage NOT amperage. [mA] and[Vh] should be set to maximum.

5.2 Conditions for mutation analysis

Since each DNA fragment has its own melting characteristic, there can be no general validparameters for mutation analysis. The following parameters have been optimized forseparating the Biometra TGGE test kit DNA (024-050). If other fragments are to be analyzed,please optimize parameters as described in chapter 3.3 and 8.

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Gel composition: 1 x TAE buffer, 8% Polyacrylamide (37,5:1), 8M Urea, 2% Glycerol

Temperature gradient: L1: 39°C, L6 54°C

Electrophoresis: 250V, 50 min

5.3 Conditions for diversity analysis of bacterial populations (geneticfingerprint)

For genetic fingerprinting primers against 16SrRNA genes are used. Depending on thecomplexity of the sample and the primer design quite a high number of PCR fragments canarise from one sample. In this regard there are two major differences to mutation analysis: 1)the number of fragments per lane is much higher 2) the amount of DNA per band is lower. Toseparate all the many fragments a rather flat gradient is used which has to be optimized verycarefully. For detecting low amounts of DNA a clear staining background is essential. Thesilver staining protocol (chapter 9.1.) has been optimized for gels with high content of ureaand formamide and thus achieves high sensitivity at low background.

For microbial diversity analysis we recommend the following conditions. However, theseparameters can only be the starting point for individual optimization.

Gel composition: 1 x TAE buffer, 6% Polyacrylamide (37,5:1), 8M Urea, 2% Glycerol, 20%Formamide

Temperature gradient: L1: 33°C, L6 44°C

Electrophoresis: 130V, 2h

5.4 Setup of electrophoresis unit

The Biometra TGGE system is a horizontal electrophoresis system. The gel is connected withthe buffer chambers by paper wicks (024-015) soaked with running buffer. To protect the gelfrom drying, it is covered with a gel cover film (024-032). The complete setup, consisting ofgel with cover film and buffer wicks, is covered with the gel cover plate (024-031). The gelcover plate has two sealings and fits precisely onto the thermoblock. It holds the buffer wicksin place and helps to build a humidity chamber around the gel. This is important to preventevaporation during the run. The setup for electrophoresis is shown in Figure 9 and Figure 10.

5.4.1 Prepare prior to assembly of the electrophoresis unit

Note: Be sure to have everything on hand, to avoid extended handling times. Do not let thedisassembled gel dry while setting up the electrophoresis system.

Prepare:

Ü parallel or perpendicular gel (see section 4)

Ü samples in loading buffer (for sample preparation see chapter 6)

Ü 500 ml 1 x TAE running buffer (chapter 10.1)

Ü 2 buffer wicks (024-015)

Ü 1 cover film (024-035)

Ü thermal coupling solution (0.01% Triton)

Ü gel cover plate (024-0321)

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5.4.2 Gel setup for electrophoresis

1) Adjust electrophoresis chamber with the 4 leveling feet.

2) Fill 250 ml running buffer in each buffer tank (check orientation of the buffer tanks:parallel or perpendicular TGGE).

Note: Wipe off any spilled buffer from the electrophoresis unit. Never run device if bufferhas been spilled.

3) Soak 2 buffer wicks with running buffer

4) Disassemble gel sandwich. Clean backside of the gel support film with a soft tissue.

5) Apply 150µl of thermal coupling solution (0.01% Triton) on the thermoblock

Note: The volume of coupling solution should be as small as possible. Excess couplingsolution leads to an irreproducible temperature distribution under the gel. The result is awavelike migration front and poor separation of fragments.

6) Place the gel on the thermoblock. The thermal coupling solution should spread overthe whole block. Avoid bubbles. Wipe off any residual coupling solution along theedges of the gel support film.

Note: The thermal coupling solution is essential for efficient heat transfer from block to gel.If bubbles are trapped under the gel support film, remove support film with gel from the blockand place it back again on the block.

7) Cover the gel with a cover film. The cover film should be placed just beneath the slots.

8) Attach pre-soaked buffer wicks on top and bottom of the gel

Gel

Cov

er f

ilm

Buffer wick

Buffer wick

Figure 9: Setup of gel, cover film and buffer wicks.

9) Load samples (approx. 5µl each for parallel gel with 8 slots, approx. 50µl forperpendicular gel with 1 slot)

Note: Be careful not to touch the samples with the buffer wick! Otherwise the samples willquickly (!) diffuse into the wick.

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10) Attach gel cover plate (the cover plate should have contact to the wicks, but shouldnot squeeze gel or wicks).

11) Close safety lid and start run.

Polybond film(024-030)

Gel

Sample

Cover plate with sealings (024-031)

Cover film(024-032)

Buffer wick(024-015)

Buffer wick(024-015)

Figure 10: Final setup for electrophoresis

5.5 Perpendicular TGGE

In perpendicular TGGE one sample is separated over a broad temperature range. Thisapplication is mainly used to check the melting behavior of a sample (see section 8.2). Forcasting of the gel use a glass plate with one large slot former (024-023). The temperaturegradient must be orientated perpendicular to the migration of the sample. The buffer tanksmust be positioned as described in Figure 11.

The migration of DNA / RNA molecules is indicated by the arrow on the safety lid of theelectrophoresis unit.

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Electrophoresis

+

Gradient

Figure 11: Positioning of buffer tanks for perpendicular TGGE. Be sure that the direction ofelectrophoresis is perpendicular to the temperature gradient. The temperature gradient is

indicated by lines L1 to L6.

5.6 Parallel TGGE

In parallel TGGE multiple samples are separated along the temperature gradient. For castingof the gel use glass plate with 32 slot formers (024-229). The buffer tanks for parallel TGGEmust be positioned as depicted in Figure 12

Gradient

Electrophoresis

+

-

Figure 12: Positioning of buffer tanks for parallel TGGE. Be sure that the direction ofelectrophoresis is parallel to the temperature gradient. The slots of the gel should be at the

same side as the markings on the block.

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6. Programming the TGGE Controller

After switching on the controller the display shows the instruments name and softwareversion. Then the main menu appears.

6.1 Software main menu

T1: 22.0°C T2: 22.0°C

Block off

A? B Elpho C programs D +

At the bottom line the display shows 4 options whichcan be retrieved by the 4 functions keys ÄA, B, C, D.

These 4 options change during programming, relativeto the chosen menu.

ÄA “?”: retrieve comments or tips about the current program step

ÄB “Elpho”: commands to open and start a program

ÄC “programs”: edit existing or create new programs

ÄD “+”: further options like printing of programs protocols, choice oflanguage, selecting signal

6.2 Creating and editing programs

Program no: __

A list B del C quit D enter

In the main menu pressing ÄC will offer the possibilityto edit a new program. First choose a program storenumber. ÄA “list” displays all program stores from 0 to99 with names or the information <empty>. ÄB “del”deletes the last entry.

After entering a number of a nonoccupied program store, thedisplay shows:

Name: > <

ABCDEFGHIJKLMNOPQRST

UVWXYZ – () α σ /, <>& +. %

A → B ABC C quit D enter

To give the program an individual name strike ÄB“ABC” to jump with the cursor into the letter field.Moving inside this field is possible by the keys ÄA“→” and ÄB “←”. Pressing ÄD “enter” the currenthigh lighted letter is stored in the name field. This stepcan be repeated 8x times. Pressing ÄD “enter” twotimes leads to the next step.

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1:L1:__ L6: alternative: T1: T2:

A ? B T1 C quit D →

Enter settings for the temperature gradient programstep:Alternatively the temperatures for L1 and L6 (firstand last thick lane on the gradient block) or for T1 andT2 (left and right edge of the gradient block) can beprogrammed. The change between programming L andT can be done by pressing ÄB "T1". If a Number has

The temperature gradient

between T1 and T2 must

not exceed 45 K.

been entered at the field L1, you have to confirm byPressing ÄD “enter”. The cursor jumps to L6. Afterprogramming L6 you will be asked "ok ?": PressingÄB "no→L1" allows new programming of L1 andL2.Pressing ÄD "yes" confirms the temperatures.

1:L1: 25.0°C L6:

Alternative:

T1: __ T2:

A ? B delete C quit D →

After entering a temperature for T1, T2, L1 or L6 thetemperature

can be Deleted by pressing ÄB "delete".

1:L1: L6:

Alternative:

T1: __ T2:

A ? B L1 C quit D →

If programming of T1 and T2 (left and right edge of thegradient block) has been done but L1 and L6 arepreferred, changing to L1 and L6 can be done bypressing ÄB "L1".

1: L1: 25.0°C L6: 60.0°C

T1: 21.1°C T2: 63.8°C

Ok ?:

A ? B no→L1 C quit D →

After setting L6 or T2 all four temperatures (L1, L6,T1, T2) are displayed.

"ok ?": Pressing ÄD "yes" leads to settingelectrophoresis parameters.

Or Pressing ÄB "no→L1" leads back to programming L1.Pressing ÄB

1: L1: 25.0°C L6: 60.0°C

T1: 21.1°C T2: 63.8°C

Ok ?:

A ? B no→T1 C quit D →

"T1" allows programming of T1 and T2. Pressing ÄB"no→T1" leads back to programming T1. Pressing ÄB"L1" allows programming of L1 and L6.

Depending on the decision of programming L1 and L6 or T1 and T2 the programmedtemperatures will be shown in the display. After programming L1 and L6 this temperatureswill be shown in the display during the next programming steps.

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1:L1: 25.0°C L6: 60.0°C

time: __0m 0s

El: 0V 500mA 30W

A ? B V*h C quit D →

Time: You can choose electrophoresis time. Standardvalues for a TGGE run are 30 – 45 min. If you enter 30and confirm by pressing ÄD “enter” you get 30 s. Ifyou enter 30 � you will get 30 min. If you enter 30 ��you will get 30 h.

1:L1: 25.0°C L6: 60.0°C

time: 30m 0s

El: 0V 500mA 30W

A ? B V*h C quit D →

V*h:

Pressing ÄB "V*h" replaces times by the more preciseVolt x hour integration.

1:L1:__ L6:

V*h: __0.00 Vh

El: 0V 500mA 30W

A ? B Time C quit D →

Pressing ÄB "Time" replaces V*H by time.

1:L1: 25.0°C L6: 60.0°C

time: 30m 0s

El: __ 0V 500mA 30W

A ? B special C quit D →

Set voltage for electrophoresis.

The TGGE controller is designed to regulate voltagebut not current or wattage. Thus current and wattagemust be set to max. values (500 mA and 30Wrespectively).

1:L1: 25.0°C L6: 60.0°C

time: 30m 0s

El: 250V 500mA 30W


Confirm voltage by pressing ÄD “enter

per ÄD “→” twice , Current and wattage are notchanged.

1: special functions

ramptime: __0m 0s

A ? B standard C quit D →

Ramptime = Ramping time

Pressing ÄB "special" gives you can set how fast theGradient block is going to the established gradient.Default setting is 1s (maximum ramping).

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1:L1: 25.0°C L6: 60.0°C

Time: 30m 0s

El: 250V 500mA 30W


2:L1:__ L2:

alternative:

T1: T2:

A ? B T1 C quit D →

Pressing ÄD “→” starts the programming of step 2 inthis program.

program no: ......

pgm end: .......step(s)

runtime: ....h.....m.......s

By pressing ÄC “quit” any change will be saved andthe following messages appear:

L1: 22.0°C L6: 22.0°C

block off


After a few seconds the main menu appears.

6.3 Start electrophoresis

Start program: __

A list B del C quit D enter

In the main menu pressing key ÄB “block” offers thepossibility to start a program.

L1: 25.0 °C L6: 60.0°C

ramp: 1 time: 0m 1s

El: 250 V 20mA 20W

A list B block C program D +

After entering a program number or choosing aprogram from the list (ÄA “list” ) the block starts toestablish the gradient. The timer for the ramping startsimmediately. The electrophoresis is started as soon asthe gradient block reaches the programmedtemperature (gradient. The limiting factor (const. V,mA or W) is indicated by an blinking arrow.)

____________________________________________ 23


L1: 25.0 °C L6: 60.0°C

hold: 1 0m 1s 0.00 Vh

El: 250 V 8mA 20W


After establishing the gradient, line two of the displaychanges. The timer now starts again and counts theelectrophoresis running time. Additionally thevolt/hour integrator starts to count.

Pressing ÄC “program” duringblock run

Program no: 4

Pgm is active!

A copy B del C quit D display

It is possible to review a program during run. Afterpressing ÄC “program” and the corresponding storenumber a warning message appears.

program no: 4

name: ................

A copy B

C quit D display

Then it is possible to display the active program (ÄD“display”) or to copy it into a new store (ÄA “copy”).It’ s not possible to change the currently runningprogram. By pressing ÄC “quit” the main menuappears again.

6.4 Stop thermo block function and electrophoresis

program: 0 TEST

Pause?

Stop?

A ? B pause C quit D stop

To stop a current running program you press ÄB“Elpho” and again ÄD “stop".

By pressing ÄC “quit” you return to previous displaywithout change.

When pressing ÄD “stop” the run will be aborted andyou leave the actual running program.

L1: 25.0 °C L6: 60.0°C

hold: 1 pause 0.00 Vh

El: 250 V 8mA 20W

A ? B Elpho C programs D +

Pressing Ä B “pause” holds the actual situation of thegradient (stopping electrophoresis, holding thetemperature gradient)“pause” alternating with “elapsedtime” is displayed.

Pressing ÄB "Elpho" (in pause status):

Pressing ÄB "contin" will continue the program

Program: 0 TEST

continue?

stop?

A ? B contin C quit D stop

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6.5 Online help [key A]

T1: 22.0°C T2: 22.0°C

block off


Pressing ÄA "?" in the main menu results in thefollowing display:

B: start/stop/pause

C: edit/delete/copy

D. special functions


L1: 25.0 °C L6: 60.0°C

Hold: 1 0m 1s 1.16Vh

El: 250 V 8mA 20W


Pressing ÄA "?" in a running program results in thefollowing display:

T1: 21.1 °C T2: 63.8°C

L1→3: 21.1 32.0 39.0

L4→6: 46.0 53.0 60.0

← = const rtime: h m

The actual temperatures of T1 and T2 as well as theactual temperatures of L1 to L6 are displayed. Thelimiting factor (const. V, mA or W) is indicated by anblinking arrow (←). The actual remainingelectrophoresis time is shown.

6.6 Options

1 print programs

2 signal

3 language

A ↑ B ↓ C quit D enter

1: Printing of program. A dot matrix printer can beconnected to the Centronics port at the controller backside.

2: Set accustic signal at the end of a program or when theprogram pauses.

3: Select between ÄA “German” or ÄB “English

4: not occupied

5: not occupied

6: not occupied

4 standard mode

5 test mode

6 void

A ↑ B ↓ C quit D enter ÄA ↑ and ÄB ↓ scrolling of display

By ÄC “quit” you return to the main menu.

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6.7 Error messages

TGGE - System

check connection

to thermoblock

TGGE connector cable is not connected to gradientblock and / or

System Controller.

Check connections!

Warning:

Gradient too large!

max. grad. T1→T2: 45°C

A ? B no→L1 C quit D enter

Programmed temperature gradient is too wide.

Or

Warning:

Gradient too large!

max. grad. T1→T2: 45°C

A ? B no→T1 C quit D enter

Program no: TEST

Pgm is active!

A copy B del C quit D display

It is possible to review a program during run. Afterpressing ÄC

Program name and store is displayed

Program no: __

Name:

not programmed!

A ↑ B ↓ C quit D enter

This program number has not been programmed.

No temperature or time has been programmed.1:L1: __ L6:

entry required

T1: T2:

A ? B L1 C quit D →

Or

1:L1: __ L6:

entry required T1: __ T2:A ? B T1 C quit D →

____________________________________________ 26


7. Sample preparation

7.1 Purity of samples

Due to the high sensitivity of the staining procedure after TGGE it is recommended to usepurified DNA, RNA or protein samples. Impurities might be misinterpreted after TGGE,thereby making the analysis of gels difficult.

PCR-amplified DNA fragments usually can be analyzed without further purification. Pleasenote, that the presence of high amounts of nonspecific, secondary PCR products may result indifficulties with interpretation of band pattern, melting profile, etc. In parallel TGGE,nonspecific bands with a higher molecular weight than the specific PCR product may bemisinterpreted as heteroduplices, or analogs with lower thermal stabilities. Therefore, prior toTGGE the PCR product should be checked in a conventional agarose gel. If necessary, purifyyour specific PCR product, e.g., by agarose gel electrophoresis and subsequent gel extraction.

7.2 Sample preparation for direct DNA analysis

1 volume of DNA/RNA samples is mixed with 1 volume of sample buffer. The resultingmixture is loaded directly on to the polyacrylamid gels. Be sure that the slots are loadedcompletely (if necessary, add 1x loading buffer to fill up the slots to maximum).

In case of low-concentration samples we recommend to prepare 5x concentrated loadingbuffer. Add 0.2 volume of this concentrated loading buffer to 0.8 volumes of the sample.

7.3 Denaturation / Renaturation for heteroduplex analysis of DNA

Mix sample with equal amount of standard DNA and heat to 95°C for 5 minutes(denaturation). Then let slowly cool down to 50°C (renaturation). This can be done byprogramming a thermocycler to 94°C for 5 minutes and then 50 °C for 15 minutes with aramping rate of –0.1°C/second. The sample is then loaded directly to the gel. In order toachieve the recommended loading volumes for diagonal or perpendicular TGGE , the samplevolume should be adjusted with running buffer.

____________________________________________ 27


8. Optimization of TGGE

There are 3 steps in the setup of a new TGGE experiment:

1) Design of the PCR fragment

2) Identification of the optimum temperature gradient

3) Parallel analysis of multiple samples

8.1 Design of DNA fragment for TGGE

The design of the DNA fragment is an important step for successful TGGE. Starting with thegene fragment of interest PCR primers should be designed with a conventional computerprogram. The melting behavior of the resulting fragment should then be checked with thePoland software. It is essential that the DNA fragment shows different melting domains. Ifthere is only one single melting domain, an artificial higher melting domain (called GCclamp) must be added during PCR.

8.1.1 Poland analysis

The melting profile of a DNA fragment can be analyzed with a computer program. ThePoland software calculates the melting behavior of a DNA fragment according to its basesequence. This software is free accessible via the internet.

(http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html ).

How to perform a Poland analysis

• Open start page (URL see above)

• 1) enter a name for the query

• 2) copy / paste DNA sequence in the sequence window

• 3) choose the Tm plot (de-activate all other plots)

• 4) submit query

• retrieve Tm plot (melting curve)

____________________________________________ 28


1) Entersequence title

2) Entersequence

3) Select graph

4) Submit

The Tm plot (second order, red color) shows the melting profile of the DNA fragmentaccording to the base sequence. The ideal fragment shows at least two distinct meltingdomains. Note that mutations can be detected in all but the highest melting domain. Thismeans that in a DNA fragment with two melting domains, mutations can only be detected inthe lower melting domain.

____________________________________________ 29


Figure 13: Tm plot of a 140bp DNA fragment resulting from Poland analysis. The secondorder curve (red color in the original) shows two different melting domains.

If the fragment consists of a single melting domain only, or if you want to scan the entirefragment for mutations, add a so called GC clamp to one end of the PCR fragment.

8.1.2 GC clamps

A GC clamp is an artificial, high melting domain which is attached to one end of the fragmentduring PCR. The name “GC clamp” implies that this short stretch will hold the DNAfragment together, preventing a dissociation into the single strands at higher temperatures.The optimum location for the GC clamp at the PCR fragment (5´ or 3´) can be easily checkedwith the Poland software. Copy / paste the GC sequence to either side of your sequence andrepeat Poland analysis. In the following box you will find different examples for a GC clamp.

short GC-clamp (23 bp): cccgc cgcgc cccgc cgccc gcc

long GC-clamp (40 bp)44: cgccc gccgc gcccc gcgcc cggcc cgccg ccccc gcccg

long GC-camp (39 bp)45: ccccg ccccc gccgc ccccc ccgcg cccgg cgccc ccgc

To integrate a GC clamp into a PCR fragment, one of the two primers has to be modified. Thenon-specific GC sequence is added to the 5´-end of the primer. Thus the GC sequence isincorporated in the fragment during PCR.

8.1.3 Chemical clamping with Psoralen

It is also possible to covalently clamp one end of a PCR fragment. To achieve this, one of theprimers carries a Psoralen molecule. Psoralen (Furo[3,2-g]coumarin, C11H6O3) is a highreactive group when exposed to UV radiation. Thus it is possible to covalently close one endof the PCR fragment. The optimal primer sequence may be 5’(Pso)pTaPpnpnp.....3’, given thepreference of Psoralen for binding between TpA and ApT pairs13,46,47. Crosslinking of thePCR product is done e.g. in a flat-bottom microtiter plate using a 365 nm UV source.Working with small volumes it may be necessary to minimize evaporation by cross-linking at4 – 10°C. The yield is not affected by temperature. The distance of the sample from the UVsource affects the yield. 15 min at 0.5 cm distance of the sample from an 8 W UV lamp issufficient.

____________________________________________ 30


8.1.4 Use of SSCP primers

In many cases primer from SSCP may be used for TGGE analysis. Nevertheless, the resultingDNA fragments should be checked in the Poland analysis. If there is only one melting domainadd a GC clamp to one of the primers (see section 8.1.2)

8.2 Identify optimum temperature gradient

Poland analysis gives the first indication, which temperature gradient should be applied forparallel analysis of multiple samples. In real life, electrophoresis is performed in the presenceof high concentrations of denaturing agents (urea and Formamide). Both lower the meltingtemperature of the DNA fragment. This allows electrophoresis at lower temperaturespreventing partial drying effects in the gel, resulting in a disturbed separation pattern.Therefore it is necessary to identify the optimum temperature gradient under experimentalconditions.

To identify the optimum temperature gradient the DNA fragment is separated in aperpendicular TGGE. This means the temperature gradient is perpendicular to the migrationdirection (see section 5.5). Thus the migration of one sample can be checked simultaneouslyat different temperatures in a single run. If the PCR fragment has been designed properly theseparation in a perpendicular temperature gradient leads to a distinct melting curve (seeFigure 14)

Figure 14: Identification of the optimum temperature gradient in a perpendicular TGGE. Atlow temperature (below T1) DNA migrates as a double strand (left side). At intermediatetemperature (between T1 and T2) the DNA opens at one side, the partial double strand is

increasingly slowed down. Above T2 the DNA separates into the single strands.

At T1 the double strand starts to melt and forms a branched structure. At T2 the partial doublestrand separates irreversibly into the single strands. Analysis of samples in parallel TGGEshould be performed precisely in this temperature range between T1 and T2.

____________________________________________ 31


8.3 Calculate temperatures at different lanes

Place the stained perpendicular gel on the TGGE thermo block showing lanes L1 to L6.Identify at what lane the double strand starts to melt (T1) and at what lane the double strandsseparates into the single strands (T2).

The calculation of the corresponding temperatures is simple, since there is a lineartemperature gradient between L1 and L6 (i.e. the temperature increment from one to the nextline is always the same).

Calculation: Divide range of gradient by five (L6 – L1), this is the temperature incrementfrom one line to the adjacent lane.

Example: calculation of temperature at line 2,5 in a temperature gradient from 40°C (L1)to 60°C (L6)

• subtract temperature at L1 from temperature L6 (range of gradient: 60-40°C = 20°C)

• divide temperature by 5 (increment per lane: 20°C/5 = 4°C)

• multiply increment by 1,5 (1,5 increments from L1 to L2,5 = 6°C)

• add this value to the temperature at L1 (40°C + 6°C)

Result: temperature at L 2,5 is 46°C

8.4 Parallel analysis of multiple samples

After identification of T1 and T2 in a perpendicular TGGE this temperature gradient is spreadover the whole block for parallel analysis.

____________________________________________ 32


T1T1

T2

Figure 15: Application of T1 and T2 in a parallel gel.

Note: The DNA fragments are separated by their melting behavior. They can be distinguishedas soon as the fragments begin to melt, i.e. they form a fork like structure (temperature higherthan T1). During electrophoresis the fragments should not separate into single strands. This isan irreversible transition resulting in diffuse bands.

Note: If there are only small differences in the migration of different samples, perform aheteroduplex analysis (see chapter 8.6)

8.5 Optimization of parallel TGGE

To improve separation in parallel TGGE the gradient should start directly at the temperaturewhere the fragments start to melt (see perpendicular gel) and should be rather flat. This meansthere should be only a moderate temperature increase over the whole gel. Different fragmentsin one sample separate as soon as the first fragment starts to melt. At a certain (higher)temperature the next fragment starts to melt. In a moderate gradient, the temperature increaseper centimeter is smaller than in a steeper gradient. This means, the distance between twotemperatures (i.e. locations in the gel) is bigger than in a steeper temperature gradient. Thisresults in a wider separation of fragments that melt at different temperatures (see Figure 16).

____________________________________________ 33


30°C

40°C

30°C

70°C

A

B

Figure 16: Parallel TGGE using a steep (A) or a flat (B) temperature gradient. With a smallertemperature gradient (30 to 40 °C, B) the separation of samples is much wider. (Note: the

temperatures in this figure are only for demonstration.)

8.6 Heteroduplex analysis with TGGE

If the difference in melting temperature between wildtype and mutant is very small,heteroduplex analysis is a rewarding approach. Heteroduplex analysis makes it very easy todistinguish between the wildtype and mutant form of a DNA fragment. The basic principle isto mix each sample with an external standard. In most cases this standard is a PCR fragmentwithout mutations, for example amplified from the wild type. After mixing the standard DNAfragment with the PCR fragment from the sample the mixture is heated and subsequentlyslowly cooled down (for protocol see section 7.3).

____________________________________________ 34


denature

re-anneal

a a AA a a A Aa aA A

Figure 17: Principle of heteroduplex analysis.The re-annealing of sample and standard results in 4 different DNA fragments. 1) Thewildtype homoduplex (AA), 2) the mutant homoduplex (aa), 3) and 4) two different

heteroduplices (Aa and aA). These heteroduplices carry at least one mismatch (disturbed basepairing) and have a significant lower melting temperature than the homoduplices.

This procedure results in a complete denaturing of both double stranded PCR fragments and asubsequent re-annealing. If the sample is different from the standard, re-annealing leads to 4different double stranded DNA fragments (see Figure 17): 1) the homoduplex of the standard(wildtype AA), 2) the homoduplex of the sample (mutant aa),3) and 4) two heteroduplicesbetween standard and sample (Aa and aA). Due to the differences between sample andstandard these heteroduplices display mismatches in their base pairing in least one position.Such mismatches have a strong impact on the melting behavior because the number of basepairs between the two strands is reduced. Therefore the heteroduplices can be easily separatedfrom the homoduplices using TGGE.

The identification of the optimum temperature gradient for the separation of a heteroduplexanalysis is absolutely the same as for a single fragment. The separation of a heteroduplexsample in a perpendicular TGGE results in 4 different melting curves. The 2 heterodupliceshave a lower melting temperature and denature at a lower temperature compared to thehomoduplices.

cold warm

elec

troph

ores

is

homoduplex aa

homoduplex AA

heteroduplex Aa

heteroduplex aA

Figure 18: Separation of a heteroduplex sample in perpendicular TGGE.

____________________________________________ 35


The temperature gradient can then be adapted in the same way as for a conventional sample(see chapter 8.2). In parallel TGGE, the samples melt as they migrate along the temperaturegradient. The heteroduplices (with mismatch) melt at a lower temperature than thehomoduplices. Thus they open earlier in the partial single strand and are slowed down in thegel matrix. The homoduplices migrate a longer distance as complete double strands and startto melt at a higher temperature (i.e. later in respect to the temperature gradient). Therefore thelower bands in parallel TGGE are the homoduplices, whereas the higher bands are theheteroduplices.

Figure 19: Schematic drawing of a screening multiple samples in a parallel TGGE. Bothhomoduplices (AA, aa) have a higher melting temperature and migrate further in the gel. The

heteroduplices melt at a lower temperature resulting in a slower migration.

8.7 Evaluation of a heteroduplex analysis

There are two possible states in heteroduplex analysis: 1) the sample is identical to thestandard (wildtype) 2) the sample is different from the wildtype. In the former case, thedenaturation / renaturation procedure results in one (the same) homoduplex. The subsequentseparation in parallel TGGE shows only a single band. In the latter case, denaturation /renaturation leads to the four different populations depicted in Figure 17. Separation inparallel TGGE results in up to four different bands (see Figure 18). If the temperature gradienthas not been correctly optimized, or if separation time was to short, there may as well only betwo or three bands.

This makes heteroduplex analysis very easy to evaluate:

number of bands result

one sample is identical to the standard

no mutation

more than one (up to 4 bands) sample is different form the standard

mutation

____________________________________________ 36


9. Staining TGGE Gels

Aside from autoradiography silver staining is the most sensitive method for detecting smallamounts of DNA, RNA or proteins in polyacrylamid gels. Other staining protocols may beused, but generally exhibit less sensitivity. This must be considered in relation to the amountof DNA loaded on the gel.

All incubation steps are done in small plastic containers which are agitated on a rockingplatform (e.g. order number 042-400 or 042-500).

Wear non-powdered protective gloves during all steps of the silver staining protocol toavoid staining artifacts due to the high sens itivity of the staining protocol.

The quality of chemicals is essential in silver staining. Prepare solutions freshly, use onlychemicals of high quality (p.a.) and fresh double distilled water.

• Important: Remove gel cover film.

• Carefully remove residual thermal coupling solution from the back of the gel (gel supportfilm) prior to staining.

• Put the polyacrylamid gel with the gel side upwards into the staining tray. Avoid airbubbles during all staining steps.

• It’s recommended to prepare at least 100 ml solution for each incubation step.

• Prepare stopping solution prior to developing.

____________________________________________ 37


9.1 Silver staining protocol

Step Time Solution

Fixation 30 min 100 ml10% glacial acid, 30% EtOH

Sensitization 2 x 10 min 100 ml30% EtOH

Washing Rinse gel 30 seconds underrunning water,

then wash 5 x 5 min Fresh aqua dest

Silver Binding 30 min 100ml 0,1 % AgNO3, prepare freshly

add 350µl Formaldehyde (37%) prior to use

Washing Rinse 30 secondsthen wash 1 minRinse again 30 seconds

Fresh aqua dest.

Developing Until bands become visible,

which can take severalminutes,

don’t let gel unattended!

Solution 1: dissolve 0,2g Sodium thiosulfate(Na2S2O3) in 10ml bidest,

Solution 2: dissolve 2,5g Sodium Carbonate(Na2CO3) in 100ml bidest

Add 100µl solution 1 to solution 2

Add 350µl Formaldehyde (37%)

Stopping discard developer

add stop solution

incubate for 30 min

Stop solution: 100 ml 10% glacial acid

Storage Up to several days at roomtemperature

10% Glycerol

Long termstorage

Remove gel from 10%glycerol and let it dry.

The gel will dry completely to the gel supportfilm and can then be stored indefinitely.

9.2 Ethidium bromide-staining

Incubate the gel in staining solution (0.5 µg/ml ethidium bromide in 1 X TBE) for 30 - 45min. Analyze under UV radiation (27).

Note: The TGGE gel must be positioned face down on the UV table. Otherwise the supportfilm will shield bands from excitation by UV light.

9.3 Autoradiography

TGGE gels can also be directly exposed to x-ray films if radiolabeled samples are analyzed.

____________________________________________ 38


Direct exposure:

Incubate the TGGE gel for 15 min. in Fixation solution (see 6.5 Silver staining). Optional:Silver stain the gel.

Remove residual buffer from the gel. Expose to an x-ray film at room temperature.

Exposure of dried TGGE gels:

Incubate the TGGE gel for 15 min. in Fixation solution (see 6.5 Silver staining). Optional:Silver stain the gel.

Incubate the gel in 2-5% glycerol for 10 minutes to prevent the gel from cracking. Incubate anappropriate sheet of cellophane (no Saran wrap!!!!!) in 2 - 5% glycerol. Layer the cellophaneon the gel. Air dry at room temperature for one day or use a gel dryer at 50°C for at least 3h.Exposure to an X-ray film.

9.4 Elution of DNA from the TGGE gel

DNA fragments which have been separated on TGGE, for example, different alleles of onegene, can be eluted from silver-stained TGGE gel and re-amplified by PCR.

Using a Pasteur pipette, puncture the gel and extract a µl piece containing the particular DNAduplex. Incubate in 20 µl TE buffer overnight. Use a 1 µl aliquot for re-amplification.

Note: This methods seems to work only if the DNA in the band has not been completelystained with silver. Therefore it is recommended to use only a short silver binding step duringthe staining protocol.

____________________________________________ 39


10. Buffers

10.1 Running buffer

Standard running buffer is 1 x TAE (Tris-Acetat EDTA, Maniatis) with a final concentrationof 0,04M Tris and 0,01M EDTA.

TAE buffer can be prepared as 50 x concentrated stock solution:

242g Tris base

57,1 ml glacial acid

100ml 0,5M EDTA (pH 8,0)

ad 1000ml bidest.

50 x TAE stocksolution

bidest total

10 x TAE 200 ml 800ml 1000 ml

1 x TAE 20 ml 980 ml 1000 ml

10.2 Sample Buffer

2x TAE

0,001% Bromphenol blue

0,001% Xylene Cyanol

0,1% Triton X100

____________________________________________ 40


11. TGGE Testkit (024-050)

The TGGE test kit was developed to get familiar with the TGGE system. It contains 3 tubeswith different DNA samples and 1 tube with loading buffer.

wild type DNA homoduplex 40 µl

Mutant DNA homoduplex 40 µl

Mutant / wildtype DNA heteroduplex 400 µl

Loading buffer 1 ml

Prior to use, samples should to be diluted as described below.

• Running buffer: 1 x TAE

• Gel composition: 8% Polyacrylamid (37,5 : 1), 8 M Urea, 1 x TAE, 2% Glycerol.

11.1 Conditions for TGGE

PERPENDICULAR TGGE PARALLEL TGGE

Samplepreparation

25 µl sample (heteroduplex)

25 µl loading buffer

2,5 µl sample (wild type, mutant orheteroduplex)

2,5 µl loading buffer

Vortex, quick spin and load 50 µl inthe broad slot

Vortex, quick spin and load 5µl each perslot

pre run temperature T1 : 20°C,

temperature T2 : 20°C

voltage 250 V

time 7 min

T1: 20°C

T2: 20°C

Voltage 250V

time 7 min

main run temperature T1 : 30°C,

temperature T2 : 70°C

voltage 250 V

time 40 min

T1: 39°C

T2: 54°C

Voltage 250V

time 50 min

____________________________________________ 41


11.2 Gel pictures TGGE test kit

perpendicular TGGE parallel TGGE

____________________________________________ 42


12. References

Note: To get an updated overview of all publications available on TGGE, please go to thewebsite of Medline (http://www.ncbi.nlm.nih.gov/PubMed/) and enter the keyword "TGGE".

1. Riesner, D., Henco, K. and Steger, G. (1990): Temperature-Gradient Gel Electrophoresis: A methodfor the analysis of conformational transitions and mutations in nucleic acids and protein. Page169-250 In Chrambach, A., Dunn, M.J., Radola, B.J.: Advances in Electrophoresis, Vol. 4, VCHVerlagsgesellschaft Weinheim

2. Kappes, S. et al.(1995): p53 mutations in ovarian tumors, detected by temperature-gradient gelelectrophoresis, direct sequencing and immunohistochemistry. Int. J. Cancer 64: 52-59

3. Milde-Langosch, K. et al. (1995): Presence and persistence of HPV and p53 mutation in cancer ofthe cervix uteri and the vulva. Int. J. Cancer 63: 639-645

4. Horn, D. et al.(1996): Three novel mutations of the NF1 gene detected by temperature gradient gelelectrophoresis of exons 5 and 8. Electrophoresis 17: 1559-1563

5. Wieland, U. et al.(1996): Quantification of HIV-1 proviral DNA and analysis of genomic diversity bypolymerase chain reaction and temperature gradient gel electrophoresis. J. Virology Methods57: 127-139

6. Kuhn, J.E. et al. (1995): Quantitation of human cytomegalovirus genomes in the brain of AIDSpatients. Journal of Medical Virology 47: 70-82

7. Linke, B. et. al. (1995): Identification and structural analysis of rearranged immunoglobulin heavychain genes in lymphomas and leukemia. Leukemia 9: 840-847.

8. Menke M.A. et al. (1995): Temperature gradient gel electrophoresis for analysis of a polymerasechain reaction-based diagnostic clonality assay in the early stages of cutaneous T-celllymphomas.

9. Hecker, R. et al. (1988): Analysis of RNA structure by temperature-gradient gel electrophoresis:viroid replication and processing. Gene 72: 59-74

10. Baumstark, T. and Riesner, D. (1995): Only one of four possible secondary structures of thecentral conserved region of potato spindle tuber viroid is a substrate for processing in a potatonuclear extract. Nucleid Acids Research 23: 4246-4254

11. Loss, P., Schmitz, M., Steger, G. and Riesner, D. (1991): Formation of a thermodynamicallymetastable structure containing hairpin II is critical for the potato spindle tuber viroid. EMBOJournal 10: 719-728

12. Riesner, D. (1998): Nucleic acid structures. In: Antisense Technology. Practical Approach Series.Oxford University Press. p1-24 (in press)

13. Wiese, U. et al. (1995): Scanning for mutations in the human prion protein open reading frame bytemporal temperature gradient gel electrophoresis. Electrophoresis 16: 1851-1860

14. Nubel, U. et al. (1996): Sequence heterogenities of genes encoding 16S rRNAs in Paenibacilluspolymyxa detected by temperature gradient gel electrophoresis.

15. Lessa, E.P. and Applebaum, G. (1993): Screening techniques for detecting allelic variation in DNAsequences. Molecular Ecology 2: 119-129

16. Richter, A., Plobner, L., Schumacher, J. 1997: Quantitatives PCR-Verfahren zur Bestimmung derPlasmidkopienzahl in rekombinanten Expressionssystemen. BIOforum 20: 545-547

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17. Henco, K. and Heibey, M. (1990): Quantitative PCR – the determination of template copy numbersby temperature gradient gel electrophoresis. Nucleic Acids Research 18: 6733-6734

18. Birmes, A. et al. (1990): Analysis of the conformational transition of proteins by temperature-gradient gel electrophoresis. Electrophoresis 11: 795-801

19. Arakawa, T. et al. (1993): Analysis of the heat-induced denaturation of proteins using temperaturegradient gel electrophoresis. Analytical Biochemistry 208: 255-259

20. Chen, X. et al. (1995): High resolution SSCP by optimization of the temperature by transverseTGGE. Nucleic Acids Research 23: 4524-4525

21. Scholz, R.B. et al. (1993): Rapid screening for Tp53 mutations by temperature gradient gelelectrophoresis: a comparison with SSCP analysis. Human Molecular Genetics 2: 2155-2158

22. Elphinstone and Baverstock, P.R. (1997): Detecting mitochondrial genotypes by temperaturegradient gel electrophoresis and heteroduplex analysis. BioTechniques 23: 982-986

23. Poland, D. (1974): Recursion relation generation of probability profiles for sequence-specificmacromolecules with long-range correlations. Biopolymers 13:1859-1871

24. Lerman, L.S. and Silverstein, K. (1987): Computational simulation of DNA-melting and itsapplication to denaturing gradient-gel electrophoresis. Meth. Enzymol. 155: 482-501

25. Steger, G. (1994): Thermal denaturation of double-stranded nucleic acids: prediction oftemperatures critical for gradient electrophoresis and polymerase chain reaction.

26. Schumacher, J, Randels, J.W. and Riesner,D. (1983): A two dimensional electrophoretictechnique for detection of circular viroids and virusoids. Anal. Biochem. 135, 288 - 295

27. Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989): Molecular cloning, Cold Spring HaborLaboratory press

28. Steger, G. and Riesner, D. (1992): Temperaturgradienten-Gelelektrophorese: eine Methode zurAnalyse von Konformationsübergängen und Mutationen in Nukleinsäuren und Proteinen. InRadola, B.J. (ed) Handbuch der elektrophorese, VCH Verlagsgesellschaft, Weinheim

29. Sheffield, V.C., Cox, D.R. and Lerman, R.M. (1989): Attachment of a 40-base-pair G+C-richsequence (GC-clamp) to genomic DNA fragments by the polymerase chain reactiob results inimproved detection of single-base changes. Proc. Natl. Acad. Sci. USA 86, 232 - 236

30. Hecker R., Wang Z., Steger G. and Riesner D. (1988): Analysis of RNA structure by temperature-gradient gel electrophoresis: viroid replication and processing. Gene 72, 59-74

31. Jiang L., Chen W., Tain L.P. and Liu Y. (1991): Temperature-gradient gel electrophoresis of applescar skin viroid. Acta Microbiol. Sin. 30, 278-283

32. Riesner D., Hecker R. and Steger G. (1988): Structure of viroid replication intermediates asstudied by thermodynamics and temperature-gradient gel electrophoresis. In Sarma R.H. andSarma M.H. (eds.) Structure & Expression, Vol. I: From Proteins to Ribosomes, Adenine press,261-285

33. Riesner D., Steger G., Zimmat R., Owens R.A., Wagenhöfer M., Hillen W., Vollbach S. and HencoK. (1989): Temperature-gradient gel electrophoresis of nuleic acids: Analysis of confor-mationaltransitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377-389

____________________________________________ 44


34. Rosenbaum V. and Riesner D. (1987): Temperature-gradient gel electrophoresis: thermodynamicanalysis of nucleic acids and proteins in purified form and in cellular extract. Biohys. Chem. 26,235-246

35. Schönborn J., Oberstraß J., Breyel E., Tittgen J., Schumacher J., Lukacs N. (1991): Monoclonallantibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts.Nucleic Acids Res. 19, 2993-3000

36. Po Tien, Steger G., Rosenbaum V., Kaper J. and Riesner D. (1987): Double-strandedcucumovirus associated RNA5: experimental analysis of nec-rogenic and non-necrogenicvariants by temperature-gradient gel electrophoresis. Nucleic Acids Res. 15, 5069-5083

37. Zimmat R., Gruner R., Hecker R., Steger G. and Riesner D. (1991): Analysis of mutations in viroidRNA by non-denaturing and temperature-gradient gel electrophoresis. In R.H. Sarma and M.H.Sarma (eds.) Structure & Methods, Vol. 3:, DNA & RNA, Adenine Press, 339-357

38. Rosenbaum V., Klahn T., Lundberg Holmgren E., von Gabain A. and Riesner D. (1992): Co-existing structures of an mRNA stability determinat: The 5‘ region of the Escherichia coli andSerratia marcescens ompA mRNA, J.Mol.Biol., in press

39. Birmes A., Sättler A., Maurer S.O. and Riesner D. (1990): „Analysis of the conformationaltransitions of proteins by temperature-gradient gel electrophoresis“. Electrophoresis 11, 795-801

40. Sättler A., Kanka S., Schrörs W. and Riesner D. (1992): „Random mutagenesis of the weakcalcium binding side in SubtilisinCarlsberg and screening for thermal stability by temperature-gradient gel electrophoresis“. Accepted for: 1st International Symposium of Subtilisin Enzymes,EMBL, Hamburg

41. Thatcher D. and Hodson B. (1981): „Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis“. Biochem. J. 197, 105-109

42. Wagenhöfer M., Hansen D. and Hillen W. (1988): „Thermal denaturation of engineered tetrepressor proteins and their complexes with tet operator and tetracycline studie by temperature-gradient gel electrophoresis“. Analytical Biochem. 175, 422-432

43. Sanguinetti C.J., Neto E.D. and Simpson A.J.G. (1994): BioTechniques 17, 915

44. Kappes, S., Milde-Langosch, K., Kressin, P., Passlack, B., Dockhorn-Dwornczak,B., Röhlke, P. and Löning, T. (1995): "p53 Mutations in ovarian tumors, detected by temperature-

gradient gel electrophoresis, direct sequencing and immunohistochemistry". Int. J. Cancer 64,52 - 59

45. Kluwe, L., MacCollin, M., Tatagiba, M., Thomas, S., Hazim, W., Haase, W. andMautner, V.-F. (1998): "Phenotypic variability associated with 14 splice-site mutations in the NF2

gene". American Journal of Medical Genetics 77, 228 - 233

46. Lerman, L. S. and Beldjord, C. (1998). "Comprehensive mutation detection withdenaturing gradient gel electrophoresis". In R.G.H. Cotton, E. Edkins and S. Forrest (eds.) Mutation

Detection, A Practical Approach, Oxford University Press, 35 - 62

47. Gamper, H., Piette, J. and Hearst, J.E. (1984): Photochem. Photobiol. 40, 29 ff

____________________________________________ 45


13. Order information

TGGE System,

Electrophoresis unit with peltier powered gradient block, free positionablebuffer chambers, integrated controller/power unit, connecting and powercables, manual, TGGE Starter Kit

024-000

TGGE Starter Kit,

1 glass plate 1 slot (024-023), 1 glass plate 8 slots (024-022), 1 glass plate 12slots (024-025), 3 bonding plates (024-021), buffer wicks (100 Stück, 024-015), 25 Polybond films (024-030), Acryl Glide sample, 3 clamps

024-003

TGGE buffer wicks 8 x 7 cm, 100 Stück 024-015

TGGE glass plate 9 x 9cm, ohne Spacer 024-021

TGGE glass plate 9 x 9cm, 1 slot, 0,5mm Spacer 024-023

TGGE glass plate 9 x 9cm, 12 slots, 0,5mm Spacer 024-025

TGGE glass plate 9 x 9cm, 18 slots, 0,5mm Spacer 024-026

TGGE glass plate 9 x 9cm, without slot, 0,5mm Spacer 024-027

TGGE Polybond Film, 8,8 x 8,8cm, 25 pcs. 024-030

TGGE Polybond Film, 8,8 x 8,8cm, 100 pcs. 024-034

TGGE cover glass plate with silicon sealings, incl. 10 cover films 7 x 6cm 024-031

TGGE cover films 7 x 6 cm, 100 pcs. 024-035

TGGE Test Kit40µl wildtype DNA, 40µl mutant DNA, 400µl heteroduplex DNA, 1mlloading buffer, manual

024-050

TGGE gel casting stand for 5 gel sandwiches 9 x 9cm 024-028

TGGE precast gels, 8,3 x 8,8 cm, 13 slots (5µl), 10 pcs. 024-040

____________________________________________ 46


14. Technical specification

TGGE System024-000

TGGE MAXI System024-200

Electrohoresis unit

Temperierung Peltier Peltier

Temperature range 5-80°C 5-80°C

Linear gradient 45 °C 45°C

Thermo block 9 x 9 cm 20 x 20 cm

Glass plates 9 x 9 cm 23,5 x 23,5 cm

Gel dimensions 7,8 x 4,2 cm 20 x 21,7 cm

Max. separation distance

Perpendicular

Parallel

4 cm

5 cm

16 cm

19 cm

Sample number (volume) 10 (5µl)

12 (3µl)

18 (1,5µl)

13* (5µl)

32 (5µl)

42* (5µl)

Dimensions (DxWxH) 23 x 23 x 23 cm 43 x 43 x 33 cm

Weight 4,2 kg 22,0 kg

* precast gels

Controller

Control of temperature gradientand electrophoresis

Control of temperature gradientand electrophoresis

Dimensions (DxWxH) 31 x 22 x 12 cm 31 x 22 x 12 cm

Weight 3,8 kg 3,5 kg

Power pack Intergrated External

Max. Voltage 400 V 400 V

Max. Amperage 500 mA 500 mA

Max. Power 30 W 50 W

Control Constant Voltage Constant Voltage

Dimensions (DxWxH) - 30 x 22 x 8 cm

Weight - 6,5 kg

____________________________________________ 47


15. Trademarks

The TGGE method is covered by patents issued to Diagen (now QIAGEN GmbH). Biometrais exclusively licensed to produce and distribute intrumentation fot perfoming TGGE.

The polymerase chain reaction (PCR) process is covered by patents issued to Hoffman-LaRoche.

Acryl-Glide is a trademark of Amresco Inc.

Biometra is a trademark of Biometra GmbH.

Whatman is a trademark of Whatman International Ltd.

The POLAND software service established by Gerhard Steger, Department of Biophysics,University of Duesseldorf, is available by internet

www.biophys.uni-duesseldorf.de/POLAND/poland.html.

____________________________________________ 48


16. EU – Konformitätserklärung, EU - Declaration of Conformity

Göttingen, den 01.10.1997

im Sinne der EG-Richtlinie über elektrische Betriebsmittel zur Verwendung innerhalbbestimmter Spannungsgrenzen 73/23/EWG, Anhang IIIfollowing the EC directive about electrical equipment for use within certain limits of voltage73/23/EWG, appendix 3

und / and

im Sinne der EG-Richtlinie für die elektromagnetische Verträglichkeit 89/336/EWG, AnhangI. following the EC directive about theelectromagnetic compability 89/336/EWG, appendix 1.

Hiermit erklären wir, daß folgende Elektrophoresegeräte:Herewith we declare that the following gel electrophoresis systems:

Typen: TGGE-System,TGGE Stromversorgungsgerät mit integriertem Controller,TGGE Elektrophoreseeinheit mit Peltier-Element,TGGE Pufferkammer

types: TGGE system,TGGE power supply with integrated controller,TGGE electrophoresis unit with Peltier element-powered gradient block,TGGE electrophoresis chamber

Best.-Nr. / Order No.: 024-000, 024-001, 024-002, 020-010, 024-090, 024-091, 024-092

den grundlegenden Anforderungen dercorresponds to the basic requirements of

EG-Niederspannungsrichtlinie 73/23 EWG i.d.F. 93/68 EWG und derEC low voltage directive 73/23 EWG in version 93/68 EWG and the

EG-Richtlinie über die elektromagnetische Verträglichkeit 89/336 EWG i.d.F. 93/68 EWGentsprechen.EC directive about the electromagnetic compatibility 89/336/EWG in version 93/68 EWG.

Folgende harmonisierte Normen wurden angewandt:The following harmonized standards have been used:

EN 50081-1 EN 50082-1EN 60555-2 EN 60555-3EN 61010-1 EN 61010-2

....................................................Quality Manager: Dr. Jürgen Otte

____________________________________________ 49


17. Instructions for return shipment

Before shipping the unit to Biometra, please read the following returninstructions.

Should you have any problems with the TGGE System, please contact your local Biometradealer or our service department:

Biometra biomedizinische Analytik GmbHService DepartmentRudolf-Wissell-Straße 30D-37079 GöttingenPhone:++49 – (0)5 51 / 50 68 6-0Fax: ++49 – (0)5 51 / 50 68 6-66

Return only defective devices. For technical problems which are not definitively recognisableas device faults please contact the Technical Service Department at Biometra.

Use the original box or a similarly sturdy one.

Label the outside of the box with “CAUTION! SENSITIVE INSTRUMENT!”

Please enclose a precise description of the fault, which also reveals during which proceduresthe fault occurred, if possible.

• Important: Clean all parts of the instrument from residues, and of biologically dangerous,chemical and radioactive contaminants. Please include a written confirmation ( use the“Equipment Decontamination Declaration” following on the next page) that the deviceis free of biologically dangerous and chemical or radioactive contaminants in eachshipment. If the device is contaminated, it is possible that Biometra will be forced torefuse to accept the device.

• The sender of the repair order will be held liable for possible losses resulting frominsufficient decontamination of the device.

• Please enclose a note which contains the following:

a) Sender’s name and address,

b) Name of a contact person for further inquiries with telephone number.

____________________________________________ 50


18. Equipment Decontamination Certificate

To enable us to comply with german law (i.e. §28 StrlSchV, §17 GefStoffV and §19 ChemG)and to avoid exposure to hazardous materials during handling or repair, will you pleasecomplete this form, prior to the equipment leaving your laboratory.

COMPANY / INSTITUTE ---_______________________________________________________________________

ADDRESS________________________________________________________________________

TEL NO ________________________ FAX NO _________________________

E-MAIL___________________________________________________________________

EQUIPMENT Model Serial No

______________ __________________

______________ __________________

If on loan / evaluation Start Date: ________ Finish Date ________

Hazardous materials used with this equipment_

__________________________________________________________________________

___________________________________________________________________________

Has the equipment been cleaned and decontaminated? YES / NO (delete)

Method of cleaning / decontamination:

__________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

NAME _____________________________ POSITION ________________________(HEAD OF DIV./ DEP./ INSTITUTE / COMPANY)

SIGNED __________________________ DATE ____________________________

PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRADISTRIBUTOR TOGETHER WITH THE EQUIPMENT.

PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING.

INSTRUMENTS NOT SHOWING THIS CERTIFICATE ATTACHED WILL BERETURNED TO SENDER.

____________________________________________ 51


19. Warranty

This Biometra instrument has been carefully built, inspected and quality controlled beforedispatch. Hereby Biometra warrants that this instrument conforms to the specificationsgiven in this manual. This warranty covers defects in materials or workmanship for 12month as described under the following conditions:

This warranty is valid for 12 months from date of shipment to the customer from Biometraor an authorized distributor. This warranty will not be extended to a third party without awritten agreement of Biometra.

This warranty covers only the instrument and all original accessories delivered with theinstrument. This warranty is valid only if the instrument is operated as described in themanual.

Biometra will repair or replace each part which is returned and found to be defective.

This warranty does not apply to wear from normal use, failure to follow operating

instructions, negligence or to parts altered or abused.

__________________________________________I


Index

1. WHAT’S NEW IN THIS MANUAL...............................................................................1

2. SAFETY INSTRUCTIONS .............................................................................................2

3. INTRODUCTION.............................................................................................................3

3.1 Principle of the method ...................................................................................................33.2 Special features of the Biometra TGGE System.............................................................43.3 Before you start: The TGGE flow chart of success ........................................................53.4 System Overview ............................................................................................................63.5 Components of the TGGE System..................................................................................7

4. PREPARING TGGE GELS.............................................................................................8

4.1 Precast gels for TGGE.....................................................................................................84.1.1 Technical specification............................................................................................84.1.2 Content ....................................................................................................................84.1.3 Rehydration of precast TGGE gels .........................................................................84.1.4 Applying precast gels on the TGGE thermoblock ..................................................8

4.2 Handcast gels for TGGE.................................................................................................94.2.1 Components of gel cuvette......................................................................................94.2.2 Treatment of glass plates.........................................................................................94.2.3 Polybond film........................................................................................................10

4.3 Assembling the glass plate sandwich............................................................................114.4 Preparing gel solution....................................................................................................124.5 Pouring gels ...................................................................................................................134.6 Disassembling the gel sandwich...................................................................................13

5. ELECTROPHORESIS WITH THE TGGE SYSTEM ...............................................14

5.1 General remarks ............................................................................................................145.2 Conditions for mutation analysis...................................................................................145.3 Conditions for diversity analysis of bacterial populations (genetic fingerprint)...........155.4 Setup of electrophoresis unit.........................................................................................15

5.4.1 Prepare prior to assembly of the electrophoresis unit ...........................................155.4.2 Gel setup for electrophoresis .................................................................................16

5.5 Perpendicular TGGE.....................................................................................................175.6 Parallel TGGE...............................................................................................................18

6. PROGRAMMING THE TGGE CONTROLLER.......................................................19

6.1 Software main menu......................................................................................................196.2 Creating and editing programs ......................................................................................196.3 Start electrophoresis ......................................................................................................226.4 Stop thermo block function and electrophoresis...........................................................236.5 Online help [key A].......................................................................................................24

_________________________________________ II


6.6 Options ..........................................................................................................................246.7 Error messages ..............................................................................................................25

7. SAMPLE PREPARATION............................................................................................26

7.1 Purity of samples...........................................................................................................267.2 Sample preparation for direct DNA analysis ................................................................267.3 Denaturation / Renaturation for heteroduplex analysis of DNA...................................26

8. OPTIMIZATION OF TGGE.........................................................................................27

8.1 Design of DNA fragment for TGGE.............................................................................278.1.1 Poland analysis......................................................................................................278.1.2 GC clamps.............................................................................................................298.1.3 Chemical clamping with Psoralen.........................................................................298.1.4 Use of SSCP primers............................................................................................30

8.2 Identify optimum temperature gradient.........................................................................308.3 Calculate temperatures at different lanes......................................................................318.4 Parallel analysis of multiple samples ............................................................................318.5 Optimization of parallel TGGE....................................................................................328.6 Heteroduplex analysis with TGGE...............................................................................338.7 Evaluation of a heteroduplex analysis...........................................................................35

9. STAINING TGGE GELS...............................................................................................36

9.1 Silver staining protocol.................................................................................................379.2 Ethidium bromide-staining............................................................................................379.3 Autoradiography............................................................................................................379.4 Elution of DNA from the TGGE gel.............................................................................38

10. BUFFERS.........................................................................................................................39

10.1 Running buffer ..........................................................................................................3910.2 Sample Buffer ...........................................................................................................39

11. TGGE TESTKIT (024-050)............................................................................................40

11.1 Conditions for TGGE................................................................................................4011.2 Gel pictures TGGE test kit........................................................................................41

12. REFERENCES................................................................................................................42

13. ORDER INFORMATION..............................................................................................45

14. TECHNICAL SPECIFICATION..................................................................................46

15. TRADEMARKS..............................................................................................................47

16. EU – KONFORMITÄTSERKLÄRUNG, EU - DECLARATION OFCONFORMITY.......................................................................................................................48

17. INSTRUCTIONS FOR RETURN SHIPMENT...........................................................49

________________________________________ III


18. EQUIPMENT DECONTAMINATION CERTIFICATE...........................................50

19. WARRANTY...................................................................................................................51

manual tgge mini 6 - cultek · the tgge system allows strictly defined linear temperature gradients...

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