Procedure No

Nachamkin FlaA Typing of Campylobacter jejuni and Campylobacter coli:

Protocols and Notes on Protocols

CAMPYNET Subgroup 3

Participant	Institute (Country)	E-mail Contact Details
Dr Bob Madden (Leader)	Queen’s University of Belfast (UK).	Bob.Madden@dardni.gov.uk
Dr Diane Newell	Veterinary Laboratories Agency (UK).	dnewell.cvl.wood@gtnet.gov.uk
*Dr Clare Harrington	*Queen’s University of Belfast (UK).	Clare.Harrington@dardni.gov.uk

The work described was undertaken at the Danish Veterinary Laboratory (DK) for this participant.

INTRODUCTION TO CAMPYNET FLAGELLIN GENE TYPING GROUP

The overall aim of the flagellin group in the first phase of CAMPYNET was to compare flagellin gene types resulting from three different protocols previously described in the literature^3,4,5,6.

· All protocols were based on polymerase chain reaction (PCR) amplification of the flagellin gene(s), followed by restriction fragment length polymorphism (RFLP) analysis of the PCR products obtained, using recommended restriction enzymes (DdeI, HinfI).

· A summary of our work to date will soon be available on the CAMPYNET website.

· We found one method to be more robust/ more discriminatory compared to the other two methods tested, and therefore advocate its use in laboratories intending to use flagellin gene typing.

RECOMMENDATION

We recommend using Nachamkin’s method^4,5 with DdeI-restriction digestion, but using a reduced anneal temperature (45 ^oC) during PCR. Protocols for this method are attached.

LIST OF ATTACHED PROTOCOLS AND REFERENCES

1. Growth Of Thermotolerant Campylobacter Strains.

2. Preparation Of Template DNA For PCR.

2.1. Phenol/ Chloroform DNA Extraction Method.

2.2. Preparation Of Cell Lysates.

3. Quantitation Of DNA.

3.1. Optical Density Estimation Of DNA Concentration.

3.2. Estimation Of DNA Concentration By Electrophoresis Against Lambda-DNA.

4. Amplification Of The FlaA Gene By Polymerase Chain Reaction (PCR).

4.1. Detection Of Amplification Products By Agarose Gel Electrophoresis.

5. Restriction Enzyme Digest Of Amplification Products; Visualisation Of Profiles By Agarose Gel Electrophoresis; Image Acquisition.

5.1. Protocol For Use With 2.5% Minigels (SVS Laboratory).

5.2. Protocol For Use With 2.0% Midigels (QUB Laboratory).

5.3. Suggestions for Acquiring Good Quality Data Suitable for Database Building

6. References.

7. Reagent Specification.

1. GROWTH OF THERMOTOLERANT CAMPYLOBACTER STRAINS

Strains should be grown for 24-72 h (typically 48 h) on non-selective 5% (v/v) horse-blood agar in a microaerobic atmosphere at 37 ^oC or 42 ^oC. Culture purity should be checked and single colonies re-streaked to ensure pure cultures are present before preparing template for PCR.

Note: Co-infection of hosts (especially pigs) by two or more Campylobacter strains has been observed previously. It is important to ensure cultures are pure before submitting to the scheme.

2. PREPARATION OF TEMPLATE DNA FOR PCR

DNA may be extracted using commercial kits, or by using standard phenol-chloroform methods, such as the method detailed in the accompanying protocols. CAMPYNET participants tested and compared two commercial kits (ISOQUICK and Easy-DNA, Invitrogen) against the given phenol-chloroform protocol. Typability of the CAMPYNET strain culture collection was not noticeably affected by the DNA extraction protocol employed.

Note: Cell lysates may be used in place of extracted DNA. This method is suited to rapid screening of large numbers of isolates in high-throughput studies, but long-term storage and re-use of lysates has been found to be problematic in our hands.

2.1. Phenol/ Chloroform DNA Extraction Method⁷

1) Sample size and cell washing: Use a 10 m l (green) loop to pick up a small ("rice-grain" sized – probably less than 1/8^th of the loop) sweep of cells from the plate. Wash cells off the loop into 1 ml SET buffer (150 mM NaCl, 15 mM EDTA, 10 mM Tris-HCl; pH 8.0) in a 1.5 ml microfuge tube, and pellet cells by centrifugation (microfuge, 3000 rpm, 5 min).

2) Cell Lysis: Resuspend the cell pellet in 570 m l SET (need a homogenous suspension without clumps of cells). Add 30 m l SDS (10% stock) and 3 m l proteinase K (20 mg/ml stock), giving a final concentration of 0.5% SDS and 100 m g/ml proteinase K. Vortex briefly, and incubate at 50 ^oC for 2hrs or overnight. No cell clumps should be visible following incubation.

3) Removal of protein: Add 600 m l of a solvent mixture of phenol: chloroform: isoamylalcohol (25:24:1), vortex for around 5-10 seconds (ensure solvents are well mixed with the lysate), then separate the phases by centrifugation (microfuge; 13000 rpm, 8 min). Transfer 550 m l of the supernatant to a new tube, add 550 m l chloroform, vortex and centrifuge as previously. Transfer 500 m l of the supernatant to a new tube.

4) Precipitation and collection of DNA: Add 1/10 volume (50 m l) 3 M sodium acetate (pH 5.3), and 2 volumes (1 ml) absolute ethanol. Precipitate at –20 ^oC for 30 min (or overnight). Pellet the DNA by centrifugation (13000 rpm, 10 min), remove the supernatant, and wash once in 500 m l of 70 % ethanol (flick tube to dislodge the pellet, then repeat the centrifugation).

5) Resuspension, RNase treatment, and solvation of DNA: Resuspend in 200 m l of TE8 buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing RNase at 10 m g/ml, and incubate for 1 hr at 37 ^oC. Allow DNA to solvate overnight at 4 ^oC, then store the DNA solution at 4 ^oC. For long term storage, preparations can be stored at -20 ^oC, but repeated freeze-thawing of the DNA is inadvisable.

Notes for individual steps:

· Phospate-buffered saline (PBS) can also be used for the initial washing stage

· The lysing incubation can be left overnight if it fits in better with the working day. The longer the incubation, the better the digestion of cellular proteins, and the easier it is to separate proteins from the DNA solution in the next step, but a 2 hr incubation is quite sufficient.

· It is important that the phenol has been equilibrated with TE8 buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.0). Equilibrated phenol: chloroform: isoamylalcohol mix (25:24:1) may be purchased from Sigma (Poole, Dorset; product code P2069). Using phenol and chloroform together improves the separation of the proteins from the rest of the lysate, but chloroform alone can be used if preferred. If phenol is used in the first solvent extraction, the chloroform-only extraction step is important to remove traces of phenol which would inhibit subsequent PCR.

· An RNase step is not essential, but incubation at 37 ^oC aids resuspension of the DNA, and it gives a cleaner DNA preparation.

2.2. Preparation of Cell Lysates

1) Harvest one 10 m l loopful of the growth and resuspend thoroughly in 500 m l of sterile distilled water in a 500 m l microfuge tube.

2) EITHER: Place tubes in the block of the Perkin-Elmer 480 Thermocycler.

a) Set the soak file to 100 ^oC, press start and close the lid.

b) When the block temperature reaches 100 ^oC, leave the tubes for 10 min.

c) Set the soak file to 4 ^oC and press start. Do not remove tubes until temperature is below 4 ^oC.

OR: Use a dri-block set to 100 ^oC, and heat for 10 min once the block has reached temperature.

3) Place tubes into a microfuge and centrifuge at 13000 rpm for 5 mins.

4) Remove supernatant (sterile pastette or 1 ml pipette) from the pelleted cell debris, and place into a fresh sterile microfuge tube.

5) The crude DNA lysates can be quantified as below, and should be stored at –20 ^oC until required.

3. QUANTITATION OF DNA

3.1. Optical Density Estimation of DNA Concentration

Optical density readings for quantitation should be taken on a UV spectrophotometer at a wavelength of 260 nm. An absorbance reading of 1.0 at 260 nm corresponds to a concentration of approximately 50 m g/ml for double stranded DNA or 40 m g/ml for single stranded DNA. The ratio between readings at 260 nm and 280 nm provides an estimation of purity of the nucleic acid. A ratio of 1.5 indicates pure DNA. Higher ratios indicate presence of RNA, whilst significantly lower values will be obtained in the presence of proteins or phenol.

1) Prepare quartz cuvettes by rinsing with 1 M NaOH, then 1 M HCl, and finally, with deionised water.

Note: Disposable plastic cuvettes (UV-specification) may also be used, in which case the above cleansing step is not necessary.

2) Zero the UV spectrophotometer against the control cuvette, containing 1000 m l TE8 buffer (or any other diluent used initially for dissolving the DNA samples).

3) Mix 5m l of each DNA solution with 995m l of TE8 buffer. The optical density of the diluted DNA is read at 260 nm in a second quartz cuvette.

4) Repeat Steps 2 and 3 at a wavelength of 280 nm.

5) Rinse the second cuvette with deionised water between samples.

6) Continue until all samples have been read.

7) Clean cuvettes as in step 1 above.

DNA Concentration Calculation

DNA Concentration (mg/ml) = A₂₆₀x 50 x dilution factor (where A₂₆₀= optical density reading at 260 nm)

Note: Units of mg/ml are equivalent to those of ng/ml.

3.2. Estimation of DNA Concentration by Electrophoresis against Lambda-DNA

This method is less accurate than spectrophotometric quantitation, but requires less sample handling, gives a reasonable estimation of DNA concentration, and has the added advantage that the quality of DNA preparation can be assessed by examination of the gel following electrophoresis. Undegraded DNA appears as a single discrete band around the same size as the Lambda-DNA, whereas degraded DNA appears as a long streak at lower molecular weights. The 1 in 10 dilutions of sample DNA prepared are frequently of an appropriate concentration to use in subsequent PCR reactions.

1) Prepare stocks of Lambda (l)-DNA in TE8 at concentrations of 50, 25, and 10 ng/ml.

Note: Ready-to-load l-stocks (loading dye included) may be prepared as described in Section 7.

2) Prepare 1 in 10 dilutions of sample DNA in TE8 buffer (eg. dilute 10 ml of DNA with 90 ml of TE8).

3) Mix 10 ml of each Lambda (l) standard and each diluted DNA with 2 ml of 6x gel loading buffer.

Note: This step is not necessary for the l-standards if using the ready-to-load l-stocks (Section 7).

4) Load 10 ml of 12 ml dye mixture for each DNA sample and l-standard onto a 1% agarose gel.

Note: If using the ready-to-load l-stocks (Section 7), 10 ml are loaded for each standard.

5) For a minigel, electrophoresis conditions are 100 V for 30 min, with 1 x TBE as running buffer.

6) Stain the gel (30 min) in 1 x TBE (or water) containing ethidium bromide at 0.5 mg/ ml.

7) Examine the gel under UV light. Estimate DNA concentration of each sample by comparison of intensity against the intensity of each of the Lambda-standards. Where DNA sample intensity is equivalent to that of the Lambda-standard, the concentration of the Lambda-standard may be taken as an estimate of the DNA concentration in the diluted DNA sample.

8) Multiply the Lambda-standard concentration from Step 7 by a factor of 10 to obtain DNA concentration in the undiluted DNA sample.

4. AMPLIFICATION OF THE FLA A GENE BY POLYMERASE CHAIN REACTION (PCR)^4,5

Primer A1: 5'-GGA TTT CGT ATT AAC ACA AAT GGT GC

Primer A2: 5'-CTG TAG TAA TCT TAA AAC ATT TTG

Recommended Modification. The published^4,5 PCR anneal temperature of 55 ^oC led to approximately 10-15 % of the CAMPYNET strains giving weak or no product. Reducing the anneal temperature to 45 ^oC significantly reduced the number of problematic strains to just 2/113. Specificity of the PCR reaction was not affected i.e. additional products were not obtained for any of the CAMPYNET strains when run at the reduced anneal temperature. It is therefore our recommendation that the PCR be performed with this reduced anneal temperature of 45 ^oC.

Note: The published PCR protocols^4,5 involve use of reagent quantities far higher than those typically employed by PCR. These may be reduced accordingly, but the effect on specificity should be thoroughly examined. Changes in Taq polymerase manufacturer also require re-evaluation of reaction conditions, since different enzymes have different requirements for magnesium ion concentration, and will therefore have different activities under the conditions described. End effects on PCR product yield should be examined, since this affects the volume of PCR product that should be used for the subsequent restriction enzyme digestion (Section 5).

· We have successfully reduced final primer concentrations from 1.0 to 0.25 mM, and Taq polymerase from 2.5 U per 100 ml reaction to 0.5 U per 50 ml reaction, with no apparent effect on product yield nor specificity for the subgroup of 36 strains tested. (Boehringer Mannheim Taq polymerase was used during this work). However, we cannot unequivocally recommend use of these modified conditions until such a time that the full set of CampyNet strains has been investigated using these modifications.

PCR Protocol

1) Dilute all DNA to ~20 ng/m l using sterile water. Dilutions can be stored at –20 ^oC until required.

2) Prepare a master mix of PCR reagents as detailed below. For N number of samples, make up sufficient cocktail for N+1 reactions. Add the Taq polymerase last, and keep on ice or in the freezer until required.

*Reagent	Supplier	Concentration	Final Concentration	Volume (m l) per reaction
Sterile, ultrapure H₂O	-	-	-	51.5
10x PCR Buffer	Gibco BRL	x10	x1	10
MgCl₂	Gibco BRL	50 mM	1.5 mM	3
dNTP mixture (dATP, dCTP, dGTP, dTTP)	Pharmacia	2 mM in each dNTP	200 m M in each dNTP	10
Primer A1	Life Technologies	10 m M	1 m M	10
Primer A2	Life Technologies	10 m M	1 m M	10
Taq polymerase	Gibco BRL	5 U/ ml	2.5 U/ 100 ml	0.5

Total Volume				95

* Composition of 10x PCR buffer will vary dependent on the manufacturer. MgCl₂may already be included in certain buffers. Attention should be paid to final MgCl₂ concentration in these buffers, and MgCl₂volume should be adjusted accordingly.

3) Dispense 95 ml of cocktail mix into each PCR tube.

4) Add 5 m l of diluted sample DNA or diluted cell lysate (both at 20 ng/m l) into each PCR tube.

5) If necessary, overlay reaction mixtures with 2 drops of mineral oil. Thermocyclers with heated lids do not require oil to be added, since the heated lid prevents condensation.

6) Place tubes into thermocycler and start the appropriate program. With the exception of the reduced anneal temperature (see above), thermal cycling conditions are taken from Nachamkin et al. (1996), as modified from Nachamkin et al. (1993):

Step Temperature (^oC) Time (sec)

1. 94 60

2. 94 15

3. 45 45

4. 72 105

No. cycles: 35

5. 72 300

Hold; 4 or 10

7) PCR reactions should be stored at 4 ^oC until ready for detection by electrophoresis.

4.1. DETECTION OF AMPLIFICATION PRODUCTS BY AGAROSE GEL ELECTROPHORESIS.

An amplicon of ~1700 bp is expected. Presence or absence of this amplicon should be checked by means of agarose gel electrophoresis and ethidium bromide staining. Different electrophoresis systems are available, and electrophoresis conditions will be affected by the system used. Details are given below for two different systems (minigel and midigel) used in our laboratories.

1) Prepare the required agarose gel ahead of time, using general purpose agarose in 1x TBE.

Minigel (7 cm wide by 10 cm long): 1 % agarose, 40 ml molten volume, two combs per gel.

Midigel (12 cm wide by 14 cm long): 2 % agarose, 100 ml molten volume, two combs per gel.

2) Add 2 ml of 6x gel loading dye to 10 m l of each PCR product.

3) Load 10 ml of each 12 ml mixture into the wells of the gel, preferably leaving outer wells empty on a midigel. Molecular weight marker (our choice is Boehringer Molecular Weight Marker VI; see Section 7) should be run at regular intervals across the gel, ideally after about every fifth sample.

4) Electrophoresis conditions (using 1 x TBE as running buffer):

Minigel: 100 V for 30 min.

Midigel: 90 V for 1.5 to 2 h.

5) Visualisation of products is using a UV transilluminator, following staining (30 min) in 1x TBE or in water containing 0.5 mg/ml ethidium bromide.

5. RESTRICTION ENZYME DIGEST OF AMPLIFICATION PRODUCTS, AND VISUALISATION OF PROFILES BY AGAROSE GEL ELECTROPHORESIS

Two methods are described below, one developed for use with a minigel system, and one developed for use with a midigel system. Methods were modified from those originally described^4,5, so as to reduce the volume of restriction enzyme used in the reaction, and to allow for the different gel systems in use in our laboratories.

Note. High concentrations of PCR reaction mix will affect the final composition of the restriction enzyme buffer, and may cause inhibition, especially for restriction enzymes working optimally in low salt buffers. Modifications to restriction enzyme reactions (enzyme or PCR product volumes) must be monitored for the ensuing possibility of incomplete digestion of products. Increased incubation times may allow further reductions in enzyme volumes used.

Note. Any changes to the gel attributes given below (dimension, composition, or volume) may affect resolution of profiles obtained under the electrophoresis conditions defined. In addition, use of data acquisition systems other than those specified may require modifications to volumes of product digested and loaded. This is due to differences in detection sensitivities.

Minigel properties: 7 cm wide by 10 cm long; 12 wells; 40 ml molten volume; 2.5% general purpose agarose in 1x TBE buffer.

Midigel properties: 12 cm wide by 14 cm long; 16-20 wells; 100 ml molten volume; 2% general purpose agarose in 1x TBE buffer.

5.1. Protocol For Use With 2.5% Minigels (SVS Laboratory)

1) Prepare a master mix of reagents as detailed below. For N number of samples, make up sufficient cocktail for N+2 reactions. Add the DdeI restriction enzyme last, and keep on ice or in the freezer until required.

Reagent	Supplier	Concentration	Final Concentration	Volume (m l) per reaction
Sterile, ultrapure H₂O	-	-	-	21.8
10x PCR Buffer (H)	Boehringer Mannheim	x10	x1	3
DdeI restriction enzyme	Boehringer Mannheim	10 U/ ml	2 U/ 30 ml	0.2
Dispensed volume				25
*(Flagellin PCR product)	-	-	-	(5)
Total reaction volume				30

*Flagellin PCR product is added to individual reaction tubes after dispensing 25 ml aliquots of the cocktail mix.

2) Dispense 25 ml of cocktail mix into each microfuge tube.

3) Add 5 m l of PCR product into each microfuge tube and mix gently.

4) Incubate tubes at 37 ^oC (water-bath preferable to heat-block) for 3 h or overnight.

5) Add 6 ml 6x gel loading dye to each reaction.

6) Store at 4 ^oC until ready for examination by electrophoresis.

7) Electrophoresis. Molecular weight marker (our choice is Promega 100 bp ladder; see Section7) should be loaded in lanes 1, 5, 8 and 12 for a 12-well minigel. Samples (16 ml of each 36 ml restriction digest/ dye mixture) are loaded into remaining wells. Electrophoresis is for 90 min at 90 V, with 1x TBE as running buffer. As a guide, the furthest dye front should be about 1 cm from the bottom of the gel.

8) Staining and Data Acquisition. Gels were stained (30 min) in 1x TBE containing 0.5 mg/ml ethidium bromide. Digital images were captured using a gel documentation system (Advanced American Biotechnology), taking care to use the same camera settings every time. Thermal images were also printed for each gel.

5.2. Protocol For Use With 2% Midigels (QUB Laboratory)

*Reagent	Supplier	Concentration	Final Concentration	Volume (m l) per reaction
Sterile, ultrapure H₂O	-	-	-	12.9
10x PCR Buffer	Life Technologies	x10	x1	2
DdeI restriction enzyme	Life Technologies	10 U/ ml	1 U/ 20 ml	0.1
Dispensed Volume				15
(Flagellin PCR product)	-	-	-	(5)
Total Volume				20

*Flagellin PCR product is added to individual reaction tubes after dispensing 25 ml aliquots of the cocktail mix.

2) Dispense 15 ml of cocktail mix into each microfuge tube.

3) Add 5 m l of PCR product into each microfuge tube and mix gently.

4) Incubate tubes at 37 ^oC (water-bath) for 2 h or overnight.

5) Add 5 ml 6x gel loading dye to each reaction.

6) Store at 4 ^oC until ready for examination by electrophoresis.

7) Electrophoresis. Leave the first and last lanes empty. Molecular weight marker (our choice is Promega 100 bp ladder; see Section 7) should be loaded in the first and last used lanes, and at regular intervals across the gel, ideally after about every third sample. Samples (entire 25 ml volume of the restriction digest/ dye mixture) are loaded into remaining wells. Electrophoresis is for 3-4 h at 90 V, with 1x TBE as running buffer.

8) Staining and Data Acquisition. Gels were stained (30 min) in water containing 0.5 mg/ml ethidium bromide. Polaroid photographs were taken (type 667) and were digitized by scanning, using a desktop digital scanner.

5.3 Suggestions for Acquiring Good Quality Data Suitable for Database Building

Molecular markers used for normalization of gels

· Our favoured molecular weight marker is the 100 bp ladder manufactured by Promega (details given in Section 7). This has a 1500 bp band, then 1000 bp down to 100 bp, in steps of 100 bp. DdeI digested products almost all fall below the 1000 bp band, and all are less than 1500 bp. A different marker may be necessary for normalization of HinfI digests (if used), since most profiles observed have one of their (typically) three bands in the least accurate region between 1500 and 1000 bp.

· The first and last loaded wells on a gel should be marker lanes, so as to allow normalization across the entire gel. With larger (midi) gels, we found it wise to leave the edge-most wells empty, due to more serious distortion effects at the edges of the gels.

· Recommendations for the frequency of loading of molecular weight markers range from every other lane as marker to every fifth lane, depending in part on the number of lanes across the gel. We did not observe any noticeable improvement in normalization when running marker in every third compared to every fourth lane.

· Analysis of profiles on a purely visual basis is not recommended long-term. It may seem quicker, but it will require additional gels to be run, whereby suspected identical profiles from different gels are run side by side to be absolutely certain of their identity.

Staining of Gels

Staining subsequent to electrophoresis was found to give more uniform staining compared to that seen for gels run with ethidium bromide included in the gel itself.

Detection and Recording of Profiles

The importance of high quality data recording is often under-estimated. It is vital for compilation of an accurate database, especially if data is to be compared between laboratories. Comparing the different methods used in our three laboratories, one was found to be far inferior at lower molecular weights compared to the other two methods. It is not our aim to recommend any particular image acquisition system, but we can provide some guidelines on assessing image quality:

· Ensure that all marker bands can be seen on the gel following staining. We have found that provided the lowest (100 bp) marker and highest (1500 bp) marker bands can be visualized, then the gel image should be of good enough contrast for subsequent analysis of restriction profiles.

· If an electronic capture system is used to acquire the gel image, ensure that no information is lost compared to simple UV visualization of the actual gel. This was a problem at one of our institutes.

· If a scanner is used for digitization of a photograph, ensure that no information is lost following the scanning procedure.

6. REFERENCES

1. Newell, D.G. 1998. A European Network to standardize and harmonize the molecular subtyping of campylobacters (CAMPYNET), pp. 583-585. In Lastovica, A., Newell, D. G., and Lastovica, E. E. (Eds.), Campylobacter IX: Proceedings of the 9^th International Workshop on Campylobacter, Helicobacter and related organisms. Institute of Child Health, University of Cape Town.

2. CAMPYNET Website. http://www.svs.dk/campynet/

3. Ayling, R.D., Woodward, M.J., Evans, S., and Newell, D.G. 1996. Restriction fragment length polymorphism of polymerase chain reaction products as applied to the differentiation of poultry campylobacters for epidemiological investigations. Res. Vet. Science 60:168-172.

4. Nachamkin, I., Bohachick, K., and Patton, C.M. 1993. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J. Clin. Microbiol. 31:1531-1536.

5. Nachamkin, I., Ung, H., and Patton, C.M. 1996. Analysis of HL and O serotypes of Campylobacter strains by the flagellin gene typing system. J. Clin. Microbiol. 34:277-281.

6. Santesteban, E., Gibson, J., and Owen, R.J. 1996. Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex. Res. Microbiol. 147:641-649.

7. Harrington, C.S., Thomson-Carter, F.M., and Carter, P.E. 1997. Evidence for recombination in the flagellin locus of Campylobacter jejuni; implications for the flagellin gene typing scheme. J. Clin. Microbiol. 35:2836-2892.

7. REAGENT SPECIFICATION

SET Buffer TE8 Buffer TBE (Tris-Borate) Buffer

150 mM NaCl 10 mM Tris-HCl 89 mM Tris-HCl

15 mM EDTA 1 mM EDTA 89 mM boric acid

10 mM Tris-HCl pH 8.0 2 mM disodium EDTA

pH 8.0 pH 8.3

2 mM dNTP Mix

Pharmacia dNTPs are provided at 100 mM. To make a 2 mM mix with respect to each dNTP:

20 ml of each dNTP (dATP, dCTP, dGTP, dTTP) plus 920 ml sterile ultrapure water

6x Gel Loading Buffer (SVS Recipe – made up in distilled water)

Component	Final concentration	Quantity per 10 ml volume
Glycerol	30%	3 g
Bromophenol blue	0.25%	25 mg (2.5 ml of 1% solution)
Xylene cyanol FF	0.25%	25 mg (2.5 ml of 1% solution)
EDTA	50 mM	1 ml of 0.5 M EDTA

6x Gel Loading Buffer (QUB Recipe – made up in 1x TBE buffer)

Component	Final concentration	Quantity per 10 ml
Sucrose	40%	4 g
Bromophenol blue	0.25%	25 mg

Making up ready-to-load marker stocks (10 ml loaded per marker lane):

Marker name*	Undiluted marker/ ml	TE/ ml	6x loading dye/ ml	Volume loaded/ ml	Undiluted Equivalent/ ml*
Boehringer VI	100	733	167	10	1.0 (250 ng)
100 bp ladder	300	533	167	10	3.0 (390 ng)
l-DNA (50 ng/ml)	200	800	200	10	2.0 (500 ng)
l-DNA (25 ng/ml)	100	900	200	10	1.0 (250 ng)
l-DNA (10 ng/ml)	40	960	200	10	0.4 (100 ng)

*l-DNA standard concentrations and undiluted equivalents given are before addition of 6x loading dye. The dilution factor incurred by addition of loading dye is compensated for by the equivalent dilution factor incurred during preparation of DNA samples for quantitation, provided the procedures in Section 3.2 are followed.

Boehringer Molecular Weight Marker VI (# 1 062 590):

Supplied at 250 ng/ml; 200 ml total

Manufacturer’s recommended loading = 1 ml undiluted marker (i.e. 250 ng) per lane.

Promega 100 bp ladder (# G210A):

Supplied at 130 ng/ml; 250 ml total.

Manufacturer’s recommended loading = 5 ml (650 ng) undiluted marker

Actually load equivalent of 3 ml undiluted marker per lane.

Boehringer Lambda DNA (# 745 782):

Supplied at 250 ng/ml.