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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
CORE SAMPLE PCR
Core Sample PCR
This protocol describes a method for re-amplifying unique PCR products from a mixture of PCR products of an initial reaction. This procedure can be useful in PCR reactions of eukaryotic genomic DNA and in reactions using degenerate oligonucleotide primers. Both of these reactions often yield a "smear" of PCR products in the correct size range, but not a discrete PCR product. Procedure
1. Perform the PCR reaction and fractionate the PCR products by Agarose gel electrophoresis (see Protocol ID#1242 and Protocol ID#1265). Separate each reaction into two samples and run each separated sample in a gel lane.
2. Cut off one lane containing one sample for each reaction from the gel with a clean scalpel and stain in Ethidium Bromide for 10 min.
3. Visualize DNA in the gel slice with UV light and cut a notch into the edge of the gel slice at the position of the migrated DNA of interest.
4. Cut off approximately 5 mm from the tip of a 200 μl pipette tip and use the larger bore pipette tip ("core sampler") to stab the Agarose in the center of the gel slice at the notched position to pickup a cylinder of Agarose containing DNA of interest (this should be approximately 10 μl of gel or so).
5. Stain the remainder of gel as in Step #2 with Ethidium Bromide and visualize the DNA in the gel with UV light to confirm that the correct regions were sampled (see Hint #2).
6. Set up and run a standard PCR reaction of 40 μl, directly adding the 10 μl core sample from Step #4 as DNA template for the reaction.
7. At the end of the reaction, either remove the samples immediately from the PCR machine and load them into the wells of a fresh Agarose gel BEFORE submerging the gel into electrophoresis buffer, or run a short program of 72°C for 5 min to melt the Agarose in the reaction and add to the wells of the Agarose gel. The PCR will solidify directly in the Agarose gel. After solidification, the gel can be submerged in running buffer (see Hint #3).
50 ng/ ml Ethidium Bromide in ddH2O (CAUTION! See Hint #1) BioReagents and Chemicals
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. It is possible to directly core-sample a gel while visualizing the DNA on a UV box; however, more than a few seconds of exposure to UV light at 254 nm wavelength will result in significant damage to the DNA and the crosslinking of the DNA to itself and the Agarose gel. The result is a template that will not efficiently yield a PCR product.
3. The reactions can be Phenol (CAUTION! See Hint #1) extracted while in the liquid phase (for example, see Protocol ID#1453) before electrophoresis or other manipulations.