book of practicals at laboratory of molecular biology
TRANSCRIPT
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CZECH UNIVERSITY OF LIFE SCIENCES PRAGUE
FACULTY OF TROPICAL AGRISCIENCES
Department of Crop Sciences and Agroforestry in Tropics and Subtropics
Laboratory of Molecular Biology
Book of practicals at
Laboratory
of molecular biology
“Plant breeding and genetic resources conservation“
Prague
2012
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Acknowledgments
This “Book of practicals at laboratory of molecular biology” was financially supported by the
project “Protocols establishment of practical exercises for molecular biology” for the subject
„Plant breeding and genetic resources conservation“ of the Fund of development of
universities FRVŠ 1940/2012.
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CONTENT
1 Safety rules and regulations in Laboratory of molecular biology
2 Preparations of Solutions
3 Methods of DNA Extraction
3.1 DNA extraction using Invisorb® Spin Plant Mini Kit (Invitek
Company)
3.2 DNA Extraction Using CTAB Method
4 ISSR Analysis
5 AFLP Analysis
6 Method of Results Processing Using Special Software
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1 Safety rules and regulations in
Laboratory of molecular biology
At the beginning of the work in the laboratory, students have to know laboratory
safety rules and regulations in Laboratory of molecular biology.
It is forbidden to eat, drink and smoke in the laboratory.
Students have to wear laboratory coats, slippers and rubber gloves.
Unauthorized experiments are strictly forbidden
The laboratory must be kept clean and organized.
Check the proper installation of the equipment. If there is any problem with equipment, do not use it and inform the supervisor.
Do not work with UV light on.
If a piece of equipment fails while being used, report it immediately to your laboratory
assistant or tutor. Never try to fix the problem yourself because you could harm yourself or the others.
Clean up your work area before leaving the laboratory.
Turn off all electric devices before leaving the laboratory.
Before leaving the laboratory, wash your hands.
Ask the supervisor if you are in doubt.
Read labels carefully.
Never “smell” a solvent directly! Read label on the solvent bottle to identify its
contents. Chemicals must never be tasted!
Check where the laboratory fire extinguisher and wash station are located and how to use them.
The staff and students are obliged to manipulate with poisonous, volatile and smelly
substances exclusively in running hood.
Always give the chemicals and reagents you used back to the place where you had taken them from.
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Special care should be taken while working with open fire, combustibles, corrosives and toxic substances.
Always inform the teacher about any accident or injury and provide the first aid if
necessary.
The reagent solutions are always casted from the reagent bottle on the unlabeled side to avoid the damage of the label. Illegible inscription and incidental substitution linked with it can cause dangerous consequences.
Concentrated acids, especially sulphuric acid, are diluted by infusion of acid into the
water. Acid is infused in the thin stream to the solution which is mixed up by the glass stick throughout the whole dilution.
Manipulation with irritating, smelling and toxic substances (i. e. chlorine, chloroform,
carbon disulphide, etc.) and easily flammable substances (i. e. gasoline, acetone, etc.) is allowed only in well aired and functional hood.
Throw toxic and nontoxic waste into the appropriate containers.
Everybody who work in the laboratory, have to respect all the rules mentioned above
and will inscribe their names into the presence book.
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2 Preparations of Solutions
Following solutions will be used in almost all protocols we are going to do during the
practicals. Thus, it is important to have them ready before beginning to work. Usually they are
already prepared in the laboratory, but if necessary, you can easily prepare them by yourself.
1. ELECTROPHORESIS
Stock solution 10x TBE1 buffer (2000 ml)
A 10x TBE buffer is prepared by dissolving 216 g Tris base in water, adding 110 g boric acid
and 18.6 g EDTA2, and bringing the final volume up to 2000 ml. The pH is adjusted to 8.3.
! The bottle with the solution has to be marked with name of solution, date and concentration.
! Do not store the solution for periods longer than 1 month.
Working solution 1x TBE buffer (1000 ml)
Dilution 10x TBE → 1x TBE buffer
Dilute 100 ml of stock solution 10x TBE buffer in 900 ml of distilled water.
An alternative to the preparation of the stock solution in the laboratory is the use of a
commercial stock solution – Rotiphorese ® Buffer 10 x TBE (Carl Roth, Germany). For the
preparation of 1 x TBE it is necessary to dilute 100 ml of 10 x TBE in 900 ml of dH2O.
2. CTAB DNA EXTRACTION METHOD
1 M TRIS (200 ml)
Weigh 24.2 g TRIS and put it into the 250 ml beaker. Add 100 ml dH2O and mix it very well.
Transfer the solution into the graduated cylinder and put the final volume of 200 ml by adding
of dH2O. Change the pH on 8.0 by using HCl. Autoclave at 120°C for 20 min and storage at
room temperature.
1 TBE = Tris-borate-EDTA buffer 2 EDTA = ethylendiaminetetraacetic acid
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0.5 EDTA (200 ml)
Weigh 29.23 g of EDTA and put it into the 250 ml beaker. Add 100 ml dH20 and mix it very
well. Change pH on 8.0 by using 10 M NaOH. Transfer the solution into the graduated
cylinder and put the final volume (200 ml) by adding of rH2O. Autoclave at 120°C for 20 min
and store at room temperature.
5 M NaCl (100 ml)
Dissolve 29.22g NaCl in 100 ml dH2O and mix it very well.
TE Buffer (100 ml)
Put 1.0 ml of 1 M TRIS (pH 8.0) into the beaker and add 0.2 ml of 0.5 M EDTA (pH 8.0) and
98.8 ml of dH2O. Mix it very well. Do not autoclave it!
CTAB Extraction buffer
Stock solution 100 ml 500 ml 1000 ml 1.4 M NaCl 5.0M 28 ml 140 ml 280 ml 20 mM EDTA 0.5M 4 ml 20 ml 40 ml 100 mM Tris pH 8.0
1.0M 10 ml 50 ml 100 ml
3% w/v CTAB 6.0% 50 ml 250 ml 500 ml Distilled water 8 ml 40 ml 80 ml
Do not autoclave extraction buffer!
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3 Methods of DNA Extraction
Both methods of the DNA extraction were optimised for the specific characteristics of yacon
(Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson) and three of its wilds relatives
(Polymnia canadensis, S. uvedalius and S. connatus). The modifications of the standard
procedures ensure high yield of clean DNA, without presence of secondary metabolites of
yacon (polyphenols, etc.), which might be a problem in following analyses.
3.1 DNA extraction using Invisorb® Spin Plant Mini Kit
(Invitek Company, Germany)
Materials and instruments:
Fresh yacon leaves, sterile gloves, autoclaved microtubes, thermoblock, pipettes, sterile tips,
source of electricity, Prefilters, Spin Filter, 2.0 ml Receiver Tubes, centrifuge, vortex
machine, mortar and pestle, spatulas, analytical scales, liquid nitrogen, container for liquid
nitrogen.
Reagents:
Lysis Buffer P, Proteinase K, Wash Buffer I, Wash Buffer II, Elution Buffer D, Binding
buffer P, liquid N2, pure EtOH, ddH2O.
WORKING PROCEDURE
At the beginning, the Proteinase K must be diluted because it is sold lyophilised. Add 1 ml of
ddH2O to the tube Proteinase K and mix thoroughly. Diluted Proteinase K must be stored at -
20°C!!
Wash Buffer I must be diluted with 30 ml of pure EtOH. Wash Buffer II must be diluted with
42 ml of pure EtOH. Tick the square indicating the adding of the EtOH on both bottles and
write down the date!
The needed amount of Elution Buffer must be pre-warmed to 65°C.
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• Homogenization of the working material
• Homogenize about 1 cm2 (the input quantity can be higher, depends on the
availability of the fresh material) of fresh leaves using a mortar and pestle under
liquid nitrogen till no large pieces of tissue remain.
• Put 60-120 mg of homogenized material into 1 ml tubes (cooled by liquid
nitrogen). (Use 120-180 mg of starting material if the starting material contains
more water.)
• Put the tubes with "plant powder" immediately in the box with liquid nitrogen.
• Put one sterile spatula into the liquid nitrogen.
• Lysis of the material
• Transfer the “plant powder” into 1.5 ml reaction tube by frozen spatula.
• Add 400 µl Lysis Buffer P and 20 µl Proteinase K and vortex briefly.
• Incubate at 65°C for 30 minutes or longer (incubation in a thermo mixer under
continuous shaking is recommended). During incubation place the Prefilter into a
2.0 ml Receiver Tube.
• Filtration of Lysis Solution and realizing optimum binding conditions
• Transfer Lysis Solution onto the Prefilter and centrifuge for 1 minute at 12 000
rpm.
• Discard the Prefilter.
• Add 200 µl of Binding Buffer P and vortex thoroughly.
• DNA Binding
• Place a new Spin Filter into a 2.0 ml Receiver Tube, transfer the suspension onto
the Spin Filter and incubate for 1 min at room temperature.
• Centrifuge at 12 000 rpm for 1 minute, discard the filtrate and place the Spin
Filter again into the 2.0 ml Receiver Tube.
• Washing I
• Add 550 µl Wash Buffer I and centrifuge at 12 000 rpm for 1 minute.
• Discard the filtrate; place the Spin Filter again into the 2.0 ml Receiver Tube.
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• Washing II
• Add 550 µl Wash Buffer II and centrifuge at 12 000 rpm for 1 minute.
• Discard the filtrate and place the Spin Filter again into the 2.0 ml Receiver Tube.
• Repeat the washing step (washing II.) once again!
• Finally discard the filtrate and centrifuge for 2 min at 12 000 rpm to remove
residual ethanol.
• Elution of the DNA
• Place the Spin Filter into a new 1.5 ml Receiver Tube and add 100 µl of the pre-
warmed Elution Buffer D3 .
• Incubate for 3 minutes (up to 10-25 minutes) at room temperature.
• Centrifuge for 1 minute at 10 000 rpm.
• Discard the Spin Filter and store the DNA at -20°C.
3 The DNA can also be eluted with a lower or a higher volume of Elution Buffer D
(depends on the expected yield of genomic DNA). But pay attention, that minimum volume for the
elution is 50 μl. If quite large amount of DNA is expected, the volume of elution can be increased
(100-200 μl).
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3.2 DNA Extraction Using CTAB Method
Materials and instruments
Yacon leaves dried in silica gel, sterile gloves, autoclaved microtubes, sterile homogenisator,
pipettes, sterile tips, thermoblock, multivortex, analytic scales, centrifuge, source of
electricity, container for liquid nitrogen.
Reagents
CTAB (pH 9), chloroform/IAA4 (24:1), isopropanol, 70% ethanol, liquid nitrogen
Before starting
Put isopropanol and 70% ethanol into the freezer. Put chloroform/IAA (24:1) into the
fridge.
Preheat a thermoblock to 65°C.
WORKING PROCEDURE
Weigh ±100 mg of yacon leaves and place it into the microtube. Put the microtube
immediately into the liquid nitrogen.
Freeze the homogenizator in liquid nitrogen (sufficient is the ending which will serve for
the tissue crushing).
Homogenize the plant tissue in the microtube using homogenizator.
Add 400 µl of CTAB (pH 9) and lightly vortex for 2 minutes.
Incubate the microtubes with plant material in a thermoblock at 65°C for cca 30-60
minutes, sometimes mix it up briefly.
Cool down the centrifuge to 4°C. Cool down the microtubes to the room temperature.
Add 150 µl of cooled chloroform/IAA (24:1) and mix it. Work in the hood!
Leave it on the blender for 10 minutes (lightly mix on the multivortex).
Centrifuge at 4°C for 5.5 minutes at the maximum speed.
Prepare new microtubes and label them.
Transfer 150 µl of the supernatant into new labelled microtubes, add 150 µl of frozen
isopropanol (work in the hood) and mix well. Put the tubes into the freezer for 5 minutes.
Centrifuge it at the maximum speed for 5 minutes.
4 IAA = Isoamyl alcohol (3-Methyl-1-butanol)
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Pipet out the supernatant (for example into the beaker) and add 200 µl of 70% ethanol into
the tubes.
Centrifuge the tubes at the maximum speed for 1 minute.
Pipet out the supernatant and dry the DNA pellets (pellets change the colour from white
into the transparent) – ±10-15 minutes.
Dissolve the pellets in the 100 µl of distilled H20 (overnight at 4°C or for 15 minutes at
65°C) or in TE at 65°C.
PURIFICATION OF DNA Reagents: 3M sodium acetate, 100 % and 70 % ethanol, redistilled water
• Put into 1.5 ml microtube 20 μl of DNA and 2 μl of sodium acetate (3M). • Add 50 μl of 100% ethanolu and mix it. • Put the microtube into the freezer with -80 °C for 30 min. • Centrifuge the microtube at 13 000 rpm for 10 min. • Pipet out the supernatant, add 500 μl of 70% ethanol and mix it. • Spin it in centrifuge. • Pour out the supernatant, dry the DNA pellet (about 15 min) and resuspend the pellet in redestilled water.
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4 ISSR Analysis Materials and instruments
Sterile gloves, autoclaved microtubes, pipettes, thermocycler, analytic scales, Erlenmayer
flask, sterile tips, electrophoresis, source of electricity, microwave own, transilluminator.
Reagents
Yacon DNA (2-5 ng/μl), PPP Master Mix with added dye (Top Bio Czech Republic), H2O
(sterile, nuclease-free, distilled), BSA5, ISSR primers (Table 3, primers which gave the best
results), agarose, 1x TBE buffer, SYBR Safe®, DNA Ladder (100 bp).
Table 3: List of ISSR primers (The University of British Columbia Biotechnology Laboratory, Canada) used. Primer Sequence Annealing
temperature
UBC 810 (GA)8T 52°C
UBC 814 (CT)8A 51°C
UBC 826 (AC)8C 54°C
UBC 834 (AG)8YT 51°C
UBC 835 (AG)8YC 48°C
UBC 841 (GA)8YC 48°C
UBC 856 (AC)8YA 52°C
Before starting
Prepare ISSR primers to the concentration 10 nmol: dilute 10 μl of ISSR primer (stock
solution) in 90 μl dH2O.
5 BSA = bovine serum albumine, serves as PCR-enhancer
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WORKING PROCEDURE
1. PCR (final volume 20 μl/one reaction)
Thaw at room temperature reagents including the DNA (or by hand or in a
thermoblock).
Centrifuge a little all vials before use.
Label each microtube by name of a sample (eventually also by the name of its owner).
If there is more than one sample, the premix (without DNA) is prepared separately to a
volume respective to the number of the samples (for each ten samples, one reaction
volume is added as a reserve).
One single reaction:
7.3 μl H2O
10 μl MasterMix (2x concentrated)
0.5 μl selected primer
0.2 μl BSA
Vortex briefly and centrifuge the premix. Divide 18 μl of the premix into the each
microtube (0.2 ml).
Add 2 μl DNA into the microtubes, shortly vortex it and briefly centrifuge them (to
get rid of air bubbles)
Put them immediately into the thermocycler and choose the right program for ISSR
primers:
1) Denaturation 94°C / 5 min
2) 40x repetitions 94°C / 1 min
specific temperature for chosen primer / 1 min
72°C / 2 min
3) Final extension 72°C / 10 min
4) Storage 4°C / forever
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2. Preparation of 2% agarose gel for electrophoresis
On the analytic scales weigh out 2 g of the agarose powder and transfer to the
Erlenmeyer flask.
Add 100 ml 1x TBE buffer to the Erlenmeyer flask.
Swirl the flask to ensure homogeneous distribution of the agarose powder and the
TBE buffer and heat the mixture in microwave oven till the liquid is completely clear
without any visible pieces of agarose.
Prepare well the gel tray with comb fixed properly.
Set aside the Erlenmeyer flask and cool it down under the tap just enough to avoid
melting of the gel tray (it must be possible to touch the forearm).
Add 1µl SYBR Safe® using a pipette and mixed well (equivalents are e.g. Ethidium
Bromide, DyeRed…).
Pour the stained liquid into the gel tray with well fixed comb.
After the gel has solidified completely (in 15-20 minutes), pull away the comb and put
whole gel into the electrophoretic bath. If it is necessary add 1x TBE buffer into the
electrophoretic bath (gel must be all covered by the buffer).
3. Electrophoresis
Leave the first well empty and put 5 μl of the samples (product of PCR)
While pipetting the sample, attention must be paid! There is a high risk of perforating
the gel by bottom of the tip.
Put 2 μl of the ladder into the first and the last well.
Cover the bath with the gel and the buffer where the wires are attached. ALWAYS
PAY ATTENTION TO THE POSITION OF THE WIRES!!!
The source should be set to 55 V and 110 mA.
Once the system is controlled to be closed well, the electricity source may be plugged
in and turned on.
Bubbles rising from the wires submerged in the buffer are the proof that the
electrophoresis works well. The electrophoresis will be left running for at least three
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hours (good to control also the position of dye added in the master mix, which first
fraction should be close to the end of the gel).
After three hours turn off the source, take the gel out and put it into the visualisation
machine.
At first, turn on the white light to fix better the gel on the transilluminator.
When the gel does not move, replace the white light by the UV light and take the
photos.
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5 AFLP Analysis
Reagents:
Yacon DNA (100-500 ng) – as concentrated as possible, all samples diluted to the same
concentration
AFLP® Core Reagent Kit (Invitrogen™, Life Technologies Czech Republic s.r.o.) (kit
components are enlisted in the Table 1),
AFLP®Pre-amp Primer Mix I (Invitrogen™, Invitrogen™, Life Technologies Czech
Republic s.r.o.), 1x TBE buffer, TE buffer, sterile dH2O, RedTaq Polymerase (Sigma Aldrich,
Czech Republic), MseI primer (5 pmol/µl), EcoRI primer (1 pmol/µl) (Table 2), dNTPs,
agarose, Ethidium Bromide, Loading Dye, DNA Ladder 100 bp.
Table 1: The components of the kit
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Table 2: List of the AFLP primers (Integrated DNA Technologies, USA).
Name of the primer Sequence (5’ – 3’)
MseI – AAA GATGAGTCCTGAGTAAAAA MseI – AAT GATGAGTCCTGAGTAAAAT MseI – AAG GATGAGTCCTGAGTAAAAG MseI – CAA GATGAGTCCTGAGTAACAA MseI – CAC GATGAGTCCTGAGTAACAC MseI - CAG GATGAGTCCTGAGTAACAG MseI – CAT GATGAGTCCTGAGTAACAT MseI – CTA GATGAGTCCTGAGTAACTA MseI – CTC GATGAGTCCTGAGTAACTC MseI – CTG GATGAGTCCTGAGTAACTG MseI - CTT GATGAGTCCTGAGTAACTT EcoRI GACTGCGTACCAATTCACG
Materials and instruments:
sterile gloves, microtubes, pipettes, thermocycler, sterile tips, analytic scales, graduated
cylinder, electrophoresis, centrifuge, vortex machine, source of electricity, container for ice,
ice, transilluminator
Before starting
First of all, the sterile gloves must be put on.
All reagents including the DNA are stored at -20°C. We will take them out and let
them thaw at room temperature. (If we are in a hurry, we can warm them up by hand
or in a thermoblock. All vials and microtubes must be centrifuged a little before use.)
!!The enzyme mix must be taken from the freezer only prior to use!!
While waiting for the reagents, prepare the PCR microtubes. They should be always
autoclaved and touched ONLY with sterile gloves.
Label each microtube by the name of a sample, eventually also by the name of its
owner.
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WORKING PROCEDURE
A. Restriction
Use the part of the kit and prepare the premix. Usually, for each 10 samples, 1 reaction
volume is added as a reserve (e.g. for 10 samples, the volume of each reagent will be
multiplied by 11).
The final volume of the reaction is 25 μl, so for one single reaction we will need:
100-500 ng of sample DNA (max. volume 18 µl)
5 μl reaction buffer
2 μl enzyme mix EcoRI/Mse I
Sterile H2O up to the final volume
Vortex the premix, centrifuged and separated into the microtubes (0.2 ml)
Add the DNA of each sample, shortly vortex the microtubes and briefly centrifuge (to
get rid of air bubbles).
Put into the thermocycler and incubate under the program:
1) Cleavage 37°C / 2 hours
2) Inactivation of the enzymes 70°C / 15 min
!!Put immediately on ice!!
B. Ligation of Adapters
Use the part of the AFLP® Core Reagent Kit I and prepare the premix (once again, for
each 10 samples, 1 reaction volume is added as a reserve). For one sample (25 µl) we
will need:
24 µl Adapter/Ligation Solution
1 µ T4 DNA Ligase
Shortly vortex the microtube and briefly centrifuged (to get rid of air bubbles).
Add directly 25 µl into the microtube from the previous step (part A. Restriction), so
the final volume of the reaction will be 50µl. Take a clean tip for each sample!!
Put the microtubes into the thermocycler and use program for the Ligation:
1) Ligation of the adapters for 2 hours at 20°C.
After the incubation, the 1:10 dilution should be performed, so take 10 µl of the R/L
product and dilute it in 90 µl of TE buffer. Such a mixture we can keep at -20°C.
To be sure that the restriction/ligation (R/L) worked well, prepare a 1.8% agarose gel
(in 1x TBE, stained with Ethidium bromide due to its better resolution capacity) and
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put about 3 µl on it dyed by 1.5 µl of Loading Dye. Put 2 µl of DNA Ladder to the
first slot of the gel. After 30 minutes (90 V) we should see a co-called “smear” on the
gel – a clear sign that our input DNA has been digested well by the restriction
enzymes.
C. Pre-selective amplification
Use a commercial kit AFLP® Pre-amp Primer Mix I and DNA polymerase (RedTaq
Polymerase, Sigma Aldrich, Czech Republic).
Prepare the premix. One single reaction will be performed in the final volume of 51
µl:
Pre-amp primer mix 40 µl
10x reaction buffer 5 µl
RedTaq DNA Polymerase 1 µl
Add 5 µl of Diluted R/L product.
Shortly vortex the microtubes and briefly centrifuged (to get rid of air bubbles).
Put the microtubes into the thermocycler and use program for the pre-selective
amplification:
1) 1x Denaturation 94°C / 2 min
2) 20x cycles: 94°C / 1 min
56°C / 30 sec
72°C / 2 min
3) Final extension 72°C / 10 min
4) Storage 4°C / forever
We can check the success of the PCR reaction by the verification of the product on the
agarose gel (as we have already done in the part B.). The result should be a “smear”
once again, but this time with some random bands (products of the pre-amp PCR
reaction).
Dilute subsequently the product 1:50 in a TE buffer (3 µl of product + 147µl of TE
buffer) and shortly vortex.
The mixture may be stored at -20°C.
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D. Selective amplification
The final volume of one reaction will be 98 µl. Please, note that we are following a
recommended protocol, in the optimal situation, as for saving the material; the
reaction volume should be 10 fold lower!!!
For one single reaction we will need:
10x Reaction buffer 10 µl
Sterile water 51 µl
MseI primer (5 pmol/µl) 5 µl
EcoRI primer (1 pmol/µl) 5 µl
dNTPs 2 µl
RedTaq Polymerase 2 µl
!!Keep the polymerase in the freezer up to the last moment!!
Add 23 µl of diluted Pre-Amp product.
Put the microtubes into the thermocycler and use program for the selective
amplification:
1) 1x 94°C / 2 min
65°C / 30 sec
72°C / 2 min
2) 8x Touch down: 94°C / 1 min
64°C / 30 sec (1°C decrease each cycle)
72°C / 1 min
3) 23x cycles: 94°C / 1 min
56°C / 30 sec
72°C / 3 min
4) Final extension 60°C / 30 min
5) Storage 4°C / forever
The product of the selective amplification step is visualised using an automatic
sequencer. The sample should be precipitated using formamide, or left such as it is and
send to the sequencing laboratory.
In the sequencing laboratory, the fluorescently labelled ladder is added (e.g.
GeneScan-ROX-500). It is the inner standard for each sample, so the length of the
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AFLP fragments can be identified and compared to other samples during different
runs of the sequencer.
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6 Method of Results Processing Using Special Software
For the processing of the results a user-friendly and not complicated software Darwin 5.0
(Perrier X. and Jacquemoud-Collet, 2006) was used.
Both methods produce dominant markers, so the best way of the evaluation is the scoring for
presence (1) or absence (0) of the band. From all the data the binary matrix is created. The
matrix is then transformed into the .txt format required by the software Darwin 5.0.
Open the Darwin 5.0
• File → Import Data Matrix
• Single data → Save Data as…
• The file will be saved with the same name as the input file, but in a format .var
• Choose “Dissimilarity”→ “Calculate From Single Data” → Upload the .var file
• “Save dissimilarity as…” → the file will be saved as .dis
• Change the number of units leaving apart two first units
• Choose “Dice” coefficient, or “Jaccard” coefficient (depends if you want similarity or
dissimilarity output) → OK
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• Choose “Tree construction” → “Neighbour Joining”
• Upload .dis file → “Save tree as…” → the file will be saved as .arb
• Tick “UnWeighted Neighbour-Joining”
• Choose “Identifiers” → Upload the .don file (was created automatically)
• Tick “Display when done” → OK
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Now we have as a result a Neighbour Joining Tree. Using the upper icons, the tree
representation might be chosen (“Tree representation”), as well as the identifiers added
(“Identification and illustration).
Identification and illustration Tree representation
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Notes