Methods: HFERP Overview and HFERP Fingerprinting Protocol (6-01-04)
This protocol is written for high-throughput analysis: for the set-up of 96 PCR reactions which are then run on 4 gels, it may be modified as needed.
Day 1
Streak 95 isolates of E. coli onto plate count agar (Difco) for single colony isolation. If E. coli are isolated into frozen stock microtiter plates, plates may be stamped for isolation. Incubate for 24 hrs at 37ºC.
Day 2
1. Add 100 µl of sterile .05 M NaOH to a sterile 96 well microtiter or PCR plate. Loop 1 µl of bacteria from 24 hr incubated PCA plates into the NaOH wash. Heat plate at 95ºC for 15 minutes in appropriate thermal cycler. Optional/Preferred: Spin down plate at 640 RPM for 10 minutes to pellet cells and concentrate DNA to the supernatant.
2. Defrost PCR reagents, mix master mix (on ice) and aliquot 23 µl of master mix into individual wells of a low profile multiplate (MJ Research) or other appropriate PCR plate, and add 2µl of the NaOH supernatant.
HFERP Master Mix
Described per reaction:
| ddiH2O | 12.65µl |
5X Gitscher buffer(*) | 5µl |
DMSO | 2.5µl |
6FAM-BOX primers(**) | 1µl |
100µM dNTP's | 1.25µl |
BSA | .2µl |
Taq Polymerase (5u/µl) | .4µl |
(*) 5X Gitscher buffer instructions are below.
(**) 1 µl of 6FAM-BOX primers consists of a mixture of 0.09 µg of unlabeled Box A1R primer per µl and 0.03 µg of 6-FAM fluorescently labeled Box A1R primer per µl (Integrated DNA Technologies, Coralville, IA).
*5X Gitschier Buffer
As found in:
Rademaker, J.L.W., F.J. Louws, and F.J. de Bruijn. 1998. Characterization
of the diversity of ecologically important microbes by rep-PCR genomic
fingerprinting. Molecular Microbial Ecology Manual 3.4.3:1-27.
Prepare and autoclave stock solutions of each reagent, and subsequently
combine them to prepare a 5X buffer. For 200 ml of 5X buffer, proportion
the stock solutions to achieve a final concentration of each of the following
reagents using sterile, double-distilled water:
To prepare 200 ml of 5X Gitschier combine:
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Final concentration
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16.6 ml of a 1 M (NH4)2SO4
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83 mM (NH4)2SO4
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67 ml of a 1 M Tris-HCl pH 8.8
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335 mM Tris-HCl pH 8.8
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6.7 ml of a 1 M MgCl2
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33.5 mM MgCl2
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1.3 ml of 1:100 dilution of a 0.5 M EDTA
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33.5 µM EDTA
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2.08 ml of a 14.4 M commercial stock of ß-mercapto-ethanol
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150 mM ß-mercapto-ethanol
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Adjust final volume to 200 ml with approximately 106 ml water.
Dispense buffer into sterile 1.5 ml microcentrifuge tubes and store at
-20° C. This buffer may be stored for several months.
3. Our project performed PCR for HFERP using an MJ Research PTC 100 (MJ Research, Waltham, MA) using the protocol specific for this thermocyclers and the Box A1R primer. PCR was initiated with an incubation at 95ºC for 2 minutes, followed by 30 cycles, consisting of 94ºC for 3 seconds, 92ºC for 30 seconds, 50ºC for 1 minute, and 65ºC for 8 minutes (40). PCR reactions were terminated after an extension at 65ºC for 8 min, and stored at 4ºC.
Day 3
1. Remove PCR reactions from the PTC and to each well add 6.6µl of a mixture of 50 µl Genescan-2500 ROX internal lane standard (Applied Biosystems, Foster City, CA) and 200 µl non-migrating loading dye (150 mg Ficoll 400 per ml, and 25 mg blue dextran per ml.) Mix dye with reaction appropriately.
2. For every 24 reactions, prepare the following:
0.5X TAE BUFFER
| ddiH2O | 1980ml |
50X TAE stock (Gibco-BRL) | 20 |
250 ml Agarose gel (Note: Pour gel when agar temp is below 60ºC to avoid comb/tray warp.)
| SeaChem LE Agarose | 3.75g |
0.5X TAE Buffer | 250ml |
3. Load 12µl for each PCR reaction to the gel, 24 reactions per gel. Gels should run at 70V for 17-18 hrs @ 4ºC with pumps attached to each gel box to recirculate buffer.
4. Obtain images from each gel as covered in Obtaining Images Using the Typhoon Scanner (6-01-04).
5. Add gel images to the Bionumerics database as covered in Adding Gel Images to the Bionumerics Database (6-01-04).
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