Can it be possible to have a PCR product from a wrong backbone? Primers of that PCR protocol were designed with respect to backbone sequences downloaded from a database. However, we realised that our backbone is not completely a match with its original sequence (we sequenced our backbone) and there are lots of point mutations on it. How can we still have a PCR product if our backbone doesn't match the sequence that we design primers from there? Can it be because of high GC % of my primers which are over 60%? Thanks in advance.
if you blast your primers against the database (Ensemble.org or UCSC genome browser) you may find lots of nearby matches to your primer sequences all over the genome of interest, with some primers more than others: you could check before hand really.. All I can suggest is increasing the annealing temperature in your PCR, make it more stringent?! or design primers which are unlikely to cross react with other genomic sequences (bit longer and check them by blasting them against the genome). All the best....
if you blast your primers against the database (Ensemble.org or UCSC genome browser) you may find lots of nearby matches to your primer sequences all over the genome of interest, with some primers more than others: you could check before hand really.. All I can suggest is increasing the annealing temperature in your PCR, make it more stringent?! or design primers which are unlikely to cross react with other genomic sequences (bit longer and check them by blasting them against the genome). All the best....
If you are amplifying from a plasmid (if this is what you mean with backbone) there is little unspecific you can amplify, unless your primers happen to be particularly bad. If your primers hit one or two mutations in the middle or the 5' side, but are fine at the 3' side, you will specifically amplify, as Maureen says, mostly because your primers rich in CG content have a low dG when they bind and tolerate better few mismatches.
Just check your primers against the sequence of your backbone and you'll find out easily if this is the case...
When you sequenced your product - how close was it to what is predicted? Increase annealing temperature - maybe stepwise and see what happens.
I deleted some of the genes by pcr out so nearly 30aa deletion that we expect,as you appreciate,we couldn't observe the deletion w/o sequencing result but we have the size of template nearly 9kb in gel and then pcr amplification seems at that position,also(with respect to no polymerase-negative pcr control reaction).
Did you try a proof reading enzyme, such as Pfu? I don't think I understand what you are trying to do. Did not realize you were amplifying plasmid! No point then to look for cross reactivity of your primers in the database.
Actually,i had changed all my template and primers after face with lot's of false positive results.My template is pcDNA3.1 plasmid, expressing channelrhodopsin enhanced yfp fused protein.I try to pcr out nearly 30-40 basepair from that fused linker region.That's why i had nearly 7kb pcr product and have problem during gel purification.But from now on,things are going well,i am on the ligation step and try to overcome low transformation efficiency.Sorry about my late response.I am gladfull for all of your interest.Best.
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