I ran the agarose gel and cut the right band, then put it to -20C, I performed PCR purification next day, but there were two bands. After two days, I ran the gel, the PCR products were almost degraded. Anyone could help me? Thank you so much.
I have seen degradation when the running buffer has been used many times and the gel has been reused and bugs have grown in the systen that chew up dna. Use new buffer for the gel and tank. The 2 bands mentioned may be that the pcr product contains a snp and under the slightly denaturing conditions of the column purification a heteroduplex forms which runs slower (larger) than the homoduplex product which runs at the expected size
Thank you so much Paul. The running buffer had been used for several times, and I put the dissloved gel liquid in two or three tubes together into 1 column. The impurity in running buffer and 60C heating, also residual chemicals in the column may cause the problem. I will perform carefully next time. Thank you very much.@Paul Rutland
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