CORE SAMPLE PCR: A method to re-PCR unique bands from products of mixed
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INTRODUCTION
PROTOCOL
- 1. Run products of a PCR amplification on 1-2% TBE agarose gel, as two or more replicate lanes.
- 2. Cut off 1 lane - flanked by marker DNA if desired, and notched to allow re-orientation with remainder of gel - and stain in preferred ethidium bromide concentration (I use 50 ng/ml for 10 min).
- 3. View excised stained piece on 254nm UV box for maximum senssitivity; notch or stab correct band(s) in sample lane.
- 4. Prepare "core samplers": using gloves and sterile scissors and cut off about 5mm from the tip of as many sterile yellow pipette tips (we use Gilson tips) as you will need for samples.
- 5. Align stained marked segment with remainder of gel. Use "core samplers" to stab out one or more cores of agarose from the centre of bands of interest, using stabbed/notched gel lane as reference: a standard gel should give about 10ul per core.
- 6. Stain remainder of gel, view and photograph at 254nm to ensure correct regions were sampled.
- 7. Use core samples as substrate in PCR reactions: I make up 40ul/reaction of reaction mix, and allow 10ul per core. Simply add core to mix, vortex a little, spin down, cover with mineral oil. PCR according to taste (not inhibited by presence of a little bromophenol blue or of 50ng/ml ethidium bromide).
- 8. At end of PCR: if you allow the tubes to cool down the reaction mix will set: 2%-odd agarose diluted 1/5 sets quite well! This is no problem for gel running as you then end the PCR on a 10 min 72oC cycle, and load the sample into wells of a gel BEFORE submerging the gel: sample will set in the wells and not float out.
- 9. If you wish to extract DNA, end at 72oC and add 50ul pre-warmed phenol / 8-OH-quinoline and vortex, add 100ul chloroform / isoamyl alcohol (24:1), vortex, spin: agarose should be in the phenol/CHCl3 phase. ALTERNATIVELY: take off mineral oil using 50ul CHCl3, take out plug of solidified sample and wash in TE, then put into 0.5ml Eppendorf-type vial with some siliconised glass wool at bottom, and a small needle hole. Put little Eppi in big Eppi without a lid, and spin 6000 rpm 10 min (a la Heery et al ., 1990; TIG 6(6):173). Collect filtrate, clean up by phenol/CHCl3 and isopropanol/ammonium acetate ppte (1 vol IP, 0.2 vol of 10M ammonium acetate).
