I amplified a 3.2kb gene from E coli K12 genomic DNA by colony PCR (1 colony in 20uL dH2O, heated at 95C for 10min, 1uL for PCR). The band seems to be of the right size (see attached colony PCR.jpg (4th lane), marker is GeneRuler, 250,500,750,1000,1500,2000,2500,3000,3500bp etc from btm to top).
To get more product for digestion and cloning, I took 0.5uL of the PCR product to be the template for a second PCR, using the same cycle, conditions and primers but with the annealing temperature raised from 50C to 55C (primers are 36-mers with 23 bases overlapping the gene). However, this gave me only two bands <1kb (2nd PCR.jpg).
For verification, I repeated the PCR again with a primer that anneals to the 1980-2000 bases in the gene and reverting to annealing at 50C. That gave me a band of ~1kb (internal primer.jpg).
I will try a few things next week but I have a few questions:
1) Why did the second PCR give only bands of low molecular weight? The major band is at 3kb but why are the low molecular weight pdts amplified instead? What could I do to use the colony PCR as the template or is this acceptable at all?
2) Would the 5C difference in annealing temperature make such a big difference? I used 50C for the colony PCR because only 23 bases anneal to the gene. With the PCR pdt, all 36bp should anneal since 55C is way lower than the Tm (~73C), shouldn't they?
3) Does the band amplified using the internal primer look too low for 1.2kb or is a little deviation ok? Is the presence of the intense band an indication that the colony PCR pdt is my gene? Or could it be a result of other non-specific pdts in the colony PCR?
4) The resolution for the higher bands are poor. Could this be due to "dead" TAE buffer?