Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
well here is a thought...
my thrid year project was based on desiging primers to identify a specific gene of a genomic DNA but i didnt order the compliment of the reverse primer, therefore i was left with two forward primers ( if that makes sense )... however the funny thing was i was able to get three different PCR products.. one of the pcr prodcuts i was able to rule out because it was bigger that target gene... however the other two pcr products could be a possibility.... i got one of the two pcr products sequenced and it turns out to be the target gene i was aiming for and this pcr product was about 400bp in lenght... after bioinformatic analysis... the only possible conclusion was that my forward primer is acting both as the reverse and the forward!!! all 23bases of the forward primer binds at one end ( so that is the forward primer) however only 7 bases bind at the down stream ( reverse primer)... the Tm of the 7bases turned out to be 32C and the annealing temp was at 55 C?? IS it possible... but interesting though.
well i suppose in away its possible... but to get very clear results i.e to get A band as oppose to multiple bands... you do need two primers forward and reverse because otherwise u will get lot of products of single stranded DNA which is not enough to be detected by gels... therefore double stranded PCR products are required.
the problem is not with ss/dsDNA, but with the quantity, which is increasing linearly in case of ssDNA and quadratically in case of dsDNA.
I could believe, you got your product, if your primer's most-3' 7-in-a-row nucleotides annealed to the reversed strand. But you should better check that before.
Cis or trans? That's what matters.
Well, the first few cycles will give very poor results indeed, but the thing is that once you start having enough amplicon from the template, those amplicon will fully match your primers, because they have included the primer at that time, and they will amplify exponentially.
I guess you have been very lucky that this 7 nt motif was specific enough, because amplification with non speicific primers, although possible, is not something very reliable. even more so when it comes to the final product. I guess a high number of cycles, a very specific forward primer, and a 7 nt motif rare enough not to ever anneal at a distance close enough to generate non specific product within your Paramaters all conspired in your favor. You are very lucky.
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
Who is online
Users browsing this forum: No registered users and 2 guests