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COMPAC-50 Comet Assay System

Von Cleaver Scientific

9963.1

Komplettsystem zur schnellen Einzelzell-Gelelektrophorese mit hohen Durchsatzraten

Platzsparendes, kompaktes System

Kammer und Zubehör zum Schutz der DNA-Proben aus schwarzem, lichtundurchlässigem Acryl

2 Objektträgerhalter zur Bearbeitung von bis zu 50 Objektträgern gleichzeitig

Zeitsparende Elektrophorese (Dauer pro Lauf ca. 20 min)

Integrierte Keramik-Kühlplatte mit Kühlakku, kein zusätzlicher Umlaufkühler nötig

1. PACKLISTE

Anz. Artikel Bezeichnung 1 Comet Assay Tank (Kammer mit Sicherheitsdeckel + integrierten Stromanschlüssen, 4 mm Stecker) 1 Paar Elektrophoresekabel 2 Objektträgerhalter (für je 25 Objektträger) 10 Behälter mit Deckel, passend für Objektträgerhalter 1 Kühlakku 1 Libelle zur Nivellierung

2. ZUBEHÖR

Objektträgerhalter (für 25 Objektträger) (1 Stück) 9964.1 Behälter für Objektträgerhalter (1 Stück) 9965.1 Behälter für Objektträgerhalter (4 Stück) 9984.1 Plus-Elektrode (1 Stück) 9966.1 Minus-Elektrode (1 Stück) 9967.1 Elektrophoresekabel Rotiphorese

® PROfessional ( 1 Paar ) 6848.1

3. TECHNISCHE DATEN

Abmessungen (B x T x H) 26,5 x 15 x 15 cm

Beladungskapazität Gerät insg. 50 Objektträger (25 x 75 mm)

Beladungskapazität pro Halter 25 Objektträger (25 x 75 mm)

Volumen 550 ml

Empfohlene Laufbedingungen 27 V / 450 mA für 20 min

Empfohlene Power Supplies Roth-STANDARD, EV2310, EV2650

BEWAHREN SIE BITTE DIE VERPACKUNG BIS ZUM ABLAUF DER GARANTIEZEIT AUF.

Weitere Informationen zum Produkt finden Sie in der anhängenden Cleaver Gebrauchsanleitung.

COMPAC-50 Comet Assay System 9963.1

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Carl Roth GmbH+Co. KG

Schoemperlenstraße 3-5 76185 Karlsruhe Postfach 100121 76231 Karlsruhe Tel.: (+49)721/5606-0 Fax: (+49)721/5606-149 E-Mail: info@carlroth.de Internet: www.carlroth.de e.d. 12/2016

COMPAC-50 Comet Assay System

By Cleaver Scientific

9963.1 Complete system for rapid high throughput single cell gel electrophoresis (SCGE)

Space-saving, compact system Chamber and accessories are made of ebony acrylic material to protect DNA samples against

light 2 slide carriers for processing of up to 50 slides simultaneously Time-saving electrophoresis (length per run ca. 20 min) Integrated ceramic cooling platform with cool pack, no additional chiller necessary

1. PACKING LIST

No. Items Description 1 Comet Assay Tank (Chamber with safety lid + integral power leads, 4 mm plugs) 1 Paar Elektrophoresis Cables 2 Slide Carrier (for 25 slides each) 10 Treatment Dishes, suitable to slide carrier 1 Cool Pack 1 Level Bubble

2. ACCESSORIES

Slide Carrier (for 25 slides) (1 carrier) 9964.1 Treatment Dish for slide carrier (1 dish) 9965.1 Treatment Dish for slide carrier (4 dishes) 9984.1 Positive Electrode (1 electr.) 9966.1 Negative Electrode (1 electr.) 9967.1 Electrophoresis Cable Rotiphorese

® PROfessional (1 pair) 6848.1

3. TECHNICAL DATA

Dimensions (W x L x H) 26,5 x 15 x 15 cm

Total Slide Capacity 50 slides (25 x 75 mm)

Slide Capacity per Rack 25 slides (25 x 75 mm)

Volume 550 ml

Recommended Running Conditions 27 V / 450 mA for 20 min

Recommended Power Supplies Roth-STANDARD, EV2310, EV2650

PLEASE RETAIN ALL PACKAGING MATERIALS UNTIL THE WARRANTY PERIOD HAS EXPIRED.

For further Information see Cleaver Instruction

Manual below.

COMPAC-50 Comet Assay System

9963.1

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Instruction Manual

COMPAC-50 High Throughput Comet Electrophoresis System

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Table of content

Page

Section 1 Safety Information

1.1 General safety Instructions 1.2 General Laboratory Safety

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6

Section 2 General Information

2.1 Introduction

2.2 Product Information 2.3 Packing List 2.4 Specifications 2.5 Care and Maintenance

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Section 3 Operating Instructions

3.1 Equipment and Materials to be Supplied by User 3.2 Reagents

3.3 Operation of Equipment

3.4 Optimization of running conditions 3.5 Related products from Cleaver Scientific 3.6 Cleaver Scientific Chilling Plate

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Section 4 Comet Assay Protocols

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Section 5 Warranty

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Section 1 Safety Information

1.1) General Safety Instructions

WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK. HOWEVER, THESE

UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY AND ARE TO BE OPERATED

ONLY BY QUALIFIED PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN THIS

INSTRUCTION MANUAL.

ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE MANUAL

THOROUGHLY.

THE UNIT MUST NEVER BE USED WITHOUT THE SAFETY LID CORRECTLY IN POSITION.

THE UNIT SHOULD NOT BE USED IF THERE IS ANY SIGN OF DAMAGE TO THE

EXTERNAL TANK OR LID.

THESE UNITS COMPLY WITH THE STATUTORY CE SAFETY DIRECTIVES: 73/23/EEC:

LOW VOLTAGE DIRECTIVE: IEC 1010-1:1990 plus AMENDMENT 1:1992

EN 61010-1:1993/BS EN 61010-1:1993

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1.2) General laboratory safety

Disposable gloves and a laboratory coat must be worn when carrying out the assay.

Safety goggles must be worn when using hazardous chemicals and where there is a

risk of reagents splashing.

All cell samples must be treated as potentially hazardous. Avoid contact with skin

and eyes.

All appropriate COSHH forms must be read prior to starting the assay, and reagent

disposal requirements must be adhered to. Care must be taken when handling

hydrochloric acid, sodium hydroxide and propidium iodide. Avoid contact with skin

and eyes.

Small amounts of cell waste can be disposed of via autoclave waste. To large

amounts of cell culture waste, add Presept disinfectant tablet, leave overnight and

dispose of down the sink following day.

Glass coverslips and slides must be disposed of in sharps bins, for incineration.

Do not pour agarose down the drains. Agarose must be disposed of via autoclave

waste.

Precautions must be taken when heating reagents in the microwave. Use heat proof

gloves.

Precautions must be taken when handling the cells. In case of spillage, clean with

70% IMS (Industrial methylated spirit).

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Section 2 General Information

2.1) Introduction

The comet assay is a process by which DNA damage (strand breaks) may be

quantified in individual cells. Under alkali conditions, other DNA damage products, known as alkali-labile sites may also be detected. Furthermore, the use of DNA repair enzymes may provide a greater degree of specificity by revealing DNA

nucleobase damage. An overview of the assay is:

1) A single cell suspension of the cells under investigation is mixed with low

melting point agarose.

2) The cell/agarose mix is layered on glass microscope slides, pre-coated with

agarose, and the agarose allowed to set.

3) The cells are lysed under high pH.

4) The presence of strand breaks and high pH allows the cellular DNA to

unwind. Electrophoresis draws the DNA out of the nucleoid body forming a

‘tail’. The amount of migration (the amount of DNA in the tail versus the

head) is proportional to the initial amount of DNA damage.

5) The high pH is neutralised by neutralization buffer and washing the slides.

6) The DNA is stained and then again washed.

7) The Comets are visualised and ‘scored’ for DNA damage assessment, using

fluorescent microscopy and image analysis software.

This process is laborious and time-consuming, having to treat every slide individually, in particular through steps 3-6. Placing the microscope slides flat, on

their largest face during electrophoresis (step 4) limits the number of slides that can fit in the electrophoresis tank.

Figure 1 COMPAC-50 High Throughput Comet Electrophoresis System

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Cleaver Scientific’s COMPAC-50 High Throughput Comet Electrophoresis System

provides a simple and effective method to process a greater number of slides simultaneously through steps 3-6 by placing the slides on their longest edge in a

microscope slide carrier (Figure 2).

Figure 2 COMPAC-50 microscope slide carrier

Coplin jars, in which lysis is performed normally in conventional comet assay carry

a maximum of 10 slides. Microscope slide carriers however can accommodate 25 slides on their side, and can be used in conjunction with treatment dishes to perform lysis, neutralisation and staining steps (steps 3,5 and 6), together with all

the associated washing steps (Figure 3). Manipulating 25 slides at a time would vastly increase the speed of the process. Also changing the conventional style of

electrophoresis tank for comet assay, to one which could accommodate the depth of the microscope slides carrier would mean slides could be transferred to the electrophoresis tank and electrophoresed whilst still in the slide carrier.

Furthermore, through using COMPAC-50 High Throughput Comet Electrophoresis System, the number of slides that can be electrophoresed is increased in a considerably smaller footprint as a standard comet tank. Decreasing the handling

of the slides will decrease the risk of possible damage or loss of LMP gels from the microscope slides, which would result in fewer comets for scoring.

Figure 3 Treatment Dishes and microscope slide carrier

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2.2) Product Information

Cleaver Scientifics’ COMPAC-50 comes as a complete package including Gel Tank

with ceramic cooling platform and cool pack, Safety Lid, 2 x 25-slidecarriers, 10x

treatment dishes and power cables. Detailed description of all the components is

written below.

Gel Tank and Lid

Ebony acrylic construction of tank and the lid ensures that nuclei remain free of exposure to background light and

potential DNA damage.

Microscope

Slide carrier

Slide carriers facilitate batch processing of multiple slides simultaneously, thus eliminating the need for manual handling of individual slides.

Treatment

Dishes

Ten treatment dishes made with ebony acrylic supplied for

batch-treatment of slides during the lysis, neutralisation, staining and washing steps.

Power Cables

Cleaver Scientifics’ power cables are designed with

protective retractable connectors which are compatible with

most power supplies.

Cooling Pack

Rapid set up cooling pack enhances the performance eliminating the need of a chiller.

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Packing List

Each unit includes the following accessories:-

2.3) Specifications

COMPAC-50

Unit Dimensions (W x L x H)

26.5 x 15 x 15cm

Total Slide Capacity

50 slides (25 x 75mm)

Slide Capacity per Rack

25

Buffer Volume

See section 3.4 *

Recommended Running Conditions

See section 3.4 *

Recommended Power Supply

CS-300V Power Pack (300V, 700mA, 150W) MIDI

*Optimization of running conditions, and volume of buffer is recommended

for every laboratory.

Comet Assay Tank and Lid

2 x 25 microscope slide carrier

10 x Treatment Dishes

Cool Pack

Power Cables

Level Bubble

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2.5) Care and Maintenance

Cleaning Large Format Vertical Units

Units are best cleaned using warm water and a mild detergent. Water at

temperatures above 600 C can cause damage to the unit and

components.

The inner module should be thoroughly rinsed with warm water or distilled

water to prevent build-up of salts but care should be taken not to damage

the enclosed electrode and vigorous cleaning is not necessary or advised. Air

drying preferably before use.

The units should only be cleaned with the following:-

Warm water with a mild concentration of soap or other mild detergent.

Compatible detergents include dishwashing liquid, Hexane and Aliphatic

hydrocarbons. The units should not be left in detergents for more than 30

minutes.

The units should never come into contact with the following cleaning

agents, these will cause irreversible and accumulative damage:-

Acetone, Phenol, Chloroform, Carbon tetrachloride, Methanol, Ethanol,

Isopropyl alcohol, Alkalis.

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Section 3 Operating Instructions

3.1) Equipment and Materials to be Supplied by User

1) AC Power Supply designed to supply constant voltage, and amperage.

Recommended output specifications are amperage to 700 mA and 21V

(constant voltage)*. The suggested power supply is Cleaver Scientific power

supply CS-300V.

*Optimization of running conditions is recommended for every laboratory,

see section 3.4.

2) Microcentrifuge

3) Vortex

4) Boiling water bath and microwave

5) 37°C water bath

6) 4°C refrigerator/cold room

7) pH meter

8) pH indicator sticks

9) Fluorescence microscope equipped with camera

10) Image and Data Analysis software

11) 20–200 μL, 200–1,000 μL pipettors

12) 200 µL, and 1000 µL Pipette tips

13) 1.5 mL microcentrifuge tubes

14) 5 mL and 10 mL stripettes

15) Pipette filler

16) 100 mL and 1 L measuring cylinders

17) Single-frosted glass microscope slides

18) 22 x 22 mm glass coverslips

19) Normal melting point Agarose

20) Low melting point Agarose

21) Propidium Iodide solution stock-1 mg/mL

22) 70% industrial methylated spirit

23) Presept disinfectant tablets

24) Double distilled (ultra-pure) water 18.2 MΩ

25) Phosphate buffered saline (PBS)

26) Na2EDTA (disodium ethylenediaminetetraacetic acid)

27) NaCl (sodium chloride)

28) NaOH (sodium hydroxide)

29) HCl (hydrochloric acid)

30) Tris-base

31) Triton X-100

32) Glass Bottles

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3.2) Reagents

LYSIS BUFFER

Stock solution: 100 mM Na2EDTA (18.6g) 2.5 M NaCl (73.05g) 10 mM Tris-base (0.6g)

Dissolve in 450 mL double distilled water Set pH to 10.0 with 10 M sodium hydroxide

Make up to 500 mL with double distilled water

Working solution: Add Triton X-100 to 1% (v/v) before use

ALKALINE ELECTROPHORESIS BUFFER (pH>13)

Stock solutions: 10 M NaOH (200 g in 500 mL) 200 mM Na2EDTA (7.4g in 100 mL)

Working solution: 300 mM NaOH, 1 mM Na2EDTA

(30 mL of 10 M NaOH + 5 mL of 200 mM Na2EDTA, make

up to 1 L with double distilled water). Ensure pH is 13.0 or above before use, using pH indicator

stick.

NOTE: Use of freshly made solution is recommended. Cool to 4°C.

NEUTRALISATION BUFFER

Working solution: 0.4 M Tris-base (4.85 g) Dissolve in 80 mL double distilled water

Set pH to 7.5 with concentrated HCl Make up to 100 mL with double distilled water

PROPIDIUM IODIDE (2.5µg/mL) Stock concentration: 1 mg/mL (1 µg/µL)

Working solution: Add 2.5 µL propidium iodide (1 mg/mL) to 1 mL double

distilled water, per slide

ICE COLD DOUBLE DISTILLED WATER

Before starting the comet assay, place 2 L double distilled water in the fridge the night before. This will be used to prepare the working solutions for the

comet assay.

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3.3) Operation of Equipment Please refer to Section 4 Day 3: Electrophoresis points 4) to 9).

3.4) Optimization of running conditions

Published comet assay results often vary extensively due to lack of standardization of protocols. Users need to optimize the conditions based on the comet assay

protocol they use in their lab. The following condition is a suggested guideline:

V/cm Voltage (V)

Maximum Current (mA)

Buffer Volume (mL)

Time (min)

1.0 21 600 550 20

0.9 19 500 550 20

0.8 17 450 550 20

0.7 15 400 550 20

The voltage should be set on constant values mentioned above (depending on the protocol the user follows). The current should be set on maximum, so the power

supply can adjust the current based on the resistance of the buffer.

The V/cm value that different laboratories use, vary between 0.7 and 1.75. If you used to use 1 V/cm on your previous tank, we strongly suggest you keep your previous condition on COMPAC-50. As long as the voltage is the same between

different runs of the comet assay, and the electrophoresis buffer is ice cold (4C), it does not matter what the current is (1).

“Any slight variation in the buffer depth along the surface may have significant effects on the voltage and also on local temperature and pH. It should be

emphasized that the total applied voltage and also the current are in theory irrelevant, since it is the voltage across the gel (approximated by the V/cm on the platform) which is the driving force for electrophoresis of the charged DNA

molecule. A higher current would hence be preferable, provided that the power supply can provide it. [Adding more buffer will in fact lead to a lower V/cm across the platform unless the total (applied) voltage is similarly increased.]” (2)

1. Godschalk RW, Ersson C, Stepnik M, Ferlinska M, Palus J, Teixeira JP, et al. Variation

of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories. Mutagenesis. 2014;29(4):241-9. 2. Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, et al. The comet assay: topical issues. Mutagenesis. 2008;23(3):143-51

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3.5) Related products from Cleaver Scientific

CS-300V CS-300V Power Supply 300V, 700mA, 150 W

CV2 Cleaver Pipette - Volume; 0.2 – 2 μL,

CV10 Cleaver Pipette - Volume; 0.5 – 10 μL,

CV20 Cleaver Pipette - Volume; 2 – 20 μL,

CV100 Cleaver Pipette - Volume; 10 – 100 μL,

CS-NRK

Rocking Shaker with 30 x 30cm platform and flat

non slip rubber mat,

CSL-

NHYBRIDBASIC

Incubator only with 2 stainless steel shelves -

110/230,

CSL-LMA50 Agarose 50g, Low melting point

CSL-CHILLPLATE Cleaver Scientific Chilling Plate – see section 3.6.

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3.6) Cleaver Scientific Chilling Plate

The Cleaver Scientific Chilling Plate (Figure 4) is constructed from aluminum

plate which accommodates 26 slides on the metal platform. Underneath there is a sliding drawer to accommodate two frozen cool packs, which provide an ice cold metal tray for rapid solidification of the low melting point agarose on

the slides. The unit is designed such that the frosted ends of the slides do not touch the metal surface, facilitating easy retrieval of the slides when the

agarose gels are solid. If there is more than one Chilling Plate in the laboratory, units are easily stackable to save space (Figure 5).

Advantages over the conventional Comet Assay ice trays:

Custom designed and manufactured precisely for Comet assay.

No need for an ice machine, and large amounts of ice.

Compact, with a smaller footprint than the conventional metal ice trays,

which sit on a large, cumbersome ice tray/bucket.

An ideal design for easy lifting of slides to overcome the problem of slides

sticking to the metal plate dues to condensation.

Figure 4 Two Chilling Plates Figure 5 Two Assembled Chilling Plates with sample slides in place

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Section 4 Comet Assay Protocols DAY 1: PREPARATION OF SLIDES

1) Pre-coat slides with 1% (w/v) normal melting point agarose in double

distilled water.

PRECAUTION: Pour agarose in 50 mL centrifuge tube and store at 37°C to prevent it from

solidifying, while coating slides. In case of solidification, discard the agarose and pour fresh agarose in tube.

2) Label the coated side with permanent marker on the bottom left corner of the

frosted section as shown below.

3) Allow to set and dry overnight at room temperature.

4) Wrap dry slides in tissue and store in plastic box provided.

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DAY 2: CELL LYSIS

All procedures are to be carried out on ice and in the dark.

1) 30,000 cells are required per treatment in duplicate (approximately 12,000 cells per gel).

2) Count 30,000 cells per treatment and transfer into a microcentrifuge tube.

3) Centrifuge at 300 x g (2,000 rpm) for 10 minutes at 4°C.

4) Meanwhile prepare 0.6% (w/v) low melting point agarose in PBS and place in

a water bath at 37°C to prevent it from setting.

5) Label the pre coated slides with name, date, and treatment using a

permanent marker.

6) Number your slides with a pencil in the corner (even permanent marker can

rub off).

7) Place the slides on a metal tray, on ice and allow slides to pre-chill for 2 minutes before adding the cells.

8) After centrifugation of the cells, discard the supernatant and disperse the pellet by vortexing. Ensure that all the supernatant has been removed from

the pellet. Place the sample tubes (containing the pelleted cells) immediately back on ice.

NOTE: When you put your sample tubes in the centrifuge, put with hinge

facing outwards. This is so the pellet will collect on this side of the

tube. Sometimes it is difficult to see the pellet and it is easy to dislodge it when removing the supernatant. Centrifuging it this way means you know where the cell pellet is going to be.

9) Resuspend the cell pellet in 200 µL 0.6% low melting point agarose and mix

up and down, without creating bubbles. Quickly transfer 80µL gel (cell and LMP agarose) onto slide. Quickly place coverslip onto gel.

10) Transfer slides into fridge and allow gels to set for 15-20 minutes.

11) Meanwhile, prepare 550 mL working solution of lysis buffer (containing 1%

triton X-100). Pour into lysis dish (Figure 3).

12) Once the gels have set, remove the cover slips and place the slides inside the

slide carrier (Figure 2) and put the slide carrier containing the slides inside the lysis dish.

13) Close the lid of the lysis dish tightly and place the lysis dish, containing the slides, in the fridge overnight at 4°C. Do not store longer than overnight (18

hours).

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DAY 3: ELECTROPHORESIS

1) Carefully remove the slide carrier containing the slides from the lysis buffer.

Take care not to disturb the gels.

2) Place the slide carrier containing the slides in the washing dish (Figure 3).

3) Wash the slides with ice cold double distilled water for 30 minutes, by adding it into the washing dish, ensuring that the slides are completely

covered. Close the lid of the washing tray to prevent DNA damage due to light.

4) Insert the pre-frozen cool packs inside the sliding drawer under the COMPAC-50 electrophoresis tank (Figure 1) to maintain optimal buffer

temperature.

5) Transfer the slide carrier containing the slides into the COMPAC-50

electrophoresis tank. Place so that the frosted ends of the slides are facing the cathode (black electrode).

6) Carefully add ice cold electrophoresis buffer to the tank. See section 3.4 for optimization of running conditions and buffer volume *.

7) Allow to sit for 20 minutes exactly. Power pack must be switched OFF during this step.

8) Carry out electrophoresis in the dark for 20 minutes. See section 3.4 for optimization of running conditions and buffer volume *.

9) Switch power pack off and carefully remove the slide carriers from the tank

and drain the electrophoresis buffer on a tissue. 10)Place the slide carrier in neutralisation dish (Figure 3) and add

neutralisation buffer to it. Close the lid and leave it for 20 minutes.

11)Remove the slide carriers from the neutralisation dish and put it in washing dish. Add ice cold double distilled water into the washing dish, close the lid and leave it for 20 minutes.

12)Remove the slide carrier containing the slides from the water and allow the slides to dry in the dark, in an incubator at 37°C overnight.

*Optimization of running conditions, and volume of buffer is recommended for every laboratory

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DAY 4: PROPIDIUM IODIDE (PI) STAINING

1) Put the slide carrier containing the slides in the washing dish, pour ice cold

double distilled water to the dish to rehydrate the slides, close the lid and leave for 30 minutes.

2) Place the slide carrier in PI labelled dish (Figure 3), in the PI work area in the laboratory.

3) Carefully add 2.5 ug/mL propidium iodide solution to the dish. Close the lid,

and incubate for 20 minutes in dark at room temperature.

4) Pour off the PI solution to the sink, and add ice cold double distilled water to

the same dish and leave for 20 minutes

5) Remove the slide carrier from the PI dish and place on tissue to drain off the

water. Leave overnight to dry in the dark, either at 37°C or room temperature.

6) Once the slides are dry, remove them from the slide carrier and store in a

slide box in the dark, until ready for image analysis.

NOTE: The slides will remain readable indefinitely and can be re-stained if

necessary.

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Section 5 Warranty

The Cleaver Scientific Ltd. (CSL) Electrophoresis units have a warranty against

manufacturing and material faults of twelve months from date of customer receipt.

If any defects occur during this warranty period, CSL will repair or replace the

defective parts free of charge.

This warranty does not cover defects occurring by accident or misuse or defects

caused by improper operation.

Units where repair or modification has been performed by anyone other than CSL

or an appointed distributor or representative are no longer under warranty from

the time the unit was modified.

Units which have accessories or repaired parts not supplied by CSL or its

associated distributors have invalidated warranty.

CSL cannot repair or replace free of charge units where improper solutions or

chemicals have been used. For a list of these please see the Care and Maintenance

subsection 2.5 pg 9.

If a problem does occur then please contact your supplier or CSL on:-

Cleaver Scientific Ltd.

Unit 4 Triton Park

Swift Valley

Brownsover Road

Rugby

CV21 1SG

Tel: +44 (0)1788 565300

Fax: +44 (0)1788 552822

Email: info@cleaverscientific.com