A method to re-PCR unique bands from products of mixed
Vernon E Coye, M Diane James, Shaon J Reid and Edward P Rybiki
The products of a PCR reaction - especially when this is done on eukaryotic
genomic DNA, and when using degenerate primers - often contain a mixture
of discrete-sized bands, one of which is the "right" one, while the others
represent products of "non-specific" priming. It can be a problem to obtain
the correct band in any state approaching purity while maintaining yield,
and attempting to purify the band by cloning all the reaction products
and then probing the library for the correct DNA can be extraordinarily
I have applied a simple "core sampling" procedure - involving "coring"
an agarose sample out of a gel, and using it as template for another round
of PCR - to get around this problem, and obtain unique bands from initially
messy backgrounds. Of course, having a visible band of the size expected
does help; however, the technique may be used on faith on "right-sized"
invisible bands if need be.
- 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
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.
NOTE: IT IS POSSIBLE TO QUICKLY CORE A STAINED GEL DIRECTLY ON A
305 OR EVEN A 254 NM UV BOX; HOWEVER, MORE THAN A FEW SECONDS OF EXPOSURE
RESULTS IN CROSS-LINKING AND NO AMPLIFICATION
- 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
- 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