PCR problem - 1.8 kb fragment amplification (Jul/16/2006 )
hi... can any of u plz suggest good enough conditions for my PCR to work... i dont have much time so cant really spend time in troubleshooting...
have isolated total RNA frm H520 cell line... have converted into cDNA using RT with oligo dT primers... the desired cDNA is 1.8kb long... now have to amplify it... problem is forward primer Tm is 77 and reverse primer Tm is 73.... please suggest conditions... thnks...
wow! how long are your primers? or, are they very GC-rich?
I have only run into such high melting temps using long primers that add sequence to the gene to clone it later...if this is what you are doing, there is a specific strategy
if they are high and you are not adding bases to your sequence, then I would suggest a 2-step PCR; so that you combine your annealing and extension into one longer step at ~70 (or better yet, gradient from 65-73 to see which is best)
but that is only a suggestion. I hope you can get it to work
i have added restriction sites at the 5' ends... total length is 30 bp each... ran a gradient PCR(60-70 degrees)... have got primer dimers in 10 out of 12... got smears in 2(lower temp)... am amplifying cDNA so tht i can clone it into a vector... primers r for the full length gene... so dont have many designing options.... help!!!!
when picking your initial Ta, consider the portion of the primers that will anneal to the original template..i.e., the part without any added bases or sites
then, set up a PCR considering that Ta (gradient is good) for ~10 cycles, followed by 20-30 with a higher Ta for specificity ( I would recommend two-step; melt at 94-98, run an anneal/extension step at 72 for a bit longer than your usual extension time, depending on your polymerase)...the later PCR steps will have product as template, allowing for the higher secondary Ta; all you are doing is making more and adding bases...this later-cycle boost in Ta will give you greater specificity after you've used the lower Ta to generate additional template...also, sometimes additives like DMSO (up to 10%, you might start with 2 or 5 to see if it helps) will give you a bit more 'flexibility' with your Ta
I would perhaps also titrate your primers down to see if you can find a range that gives you better product and gets rid of some of your dimers?
anyways, hope this helps