We are mapping using PCR-amplified SSLP markers between Col and LER
ecotypes. Our problem is in detecting small differences between small
sized fragments (e.g. 114 bp with a difference of only 4 bp).
The PCR reactions conditions are standard: genomic DNA, 5pM of each
primer, 200uM dNTP"s, 50mM KCl, 10mM Tris-HCl pH 9, 2mM Mg++, 0.01%
gelatin, 0.1% Triton X-100 and 2 units of TDP in 20ul reactions. Cycling
conditions are 94C for 15 seconds, 55C for 15 seconds, and 72C for 30
seconds X40 cycles.
The products have been run out on 8% non-denaturing polyacrylamide gels
and 2% SMW agarose in 1XTBE. The bands on the PAGE gel are wavy and
very uneven, making reliable fragment size assessment difficult. Could
the detergent or other components in the PCR product be affecting the
mobility of the fragments, and if so, is there a quick and cheap
clean-up protocol for the PCR product? On the agarose gels, the same
products show even bands, but resolution of small size differences is
not as clear.
We would appreciate any advice on the best protocol for band resolution.