Hi, I am using the same DNA template to run two 7kb PCR experiments with the same cycling conditions simultaneously. The only difference between the two experiments is the primer pair. One result shows no band whereas the other result shows a smear. What could be the issue? Thanks.
perhaps the annealing temperature is too high and the smear is one primer annealing and no amplification is both primers not annealing. Are you getting a primer dimer diffuse band at about 100 bases. More details are needed to get an answer that is more than guesswork
Could be one or more of several issues. A smear indicates non-specific amplification.
Do you have positive controls? Did they work? How about your negative controls?
Can you run the amplifications separately? The pairs of primers may have different annealing temps.
7 kb is a larger than typical amplicon, but size alone isn't a reason for no amplification. How long is your extension step?
Thanks for replying. I focused on running just one set of primers.
I tried lowering the temp but that introduced primer dimers.
Attached is a picture of the faint smear using an annealing temperature of 60 degrees as recommended by the manual.
Extension for 2 and half minutes as the manual recommends 15-30sec per kb.
thanks.
I would check the OD260/280 of your template dna to make sure that it is good quality. When a long pcr is not working I would give the extension longer than the ideal. So in your case 5 minute extension time and a 5 minute initial denaturation time and maybe shorten the extension time when it works. Just in case your template has high GC or high AT regions use the high GC buffer if your polymerase comes with a high GC buffer. If not then try running the pcr in 1M betaine to help template melting. What are the calculated melting temperatures for your primers?
Thank you, I'll try a longer extension time.
Tm of primers are 54 and 57 degrees. I tried annealing temp of 57 degrees and that introduced primer dimers.
what can I do if the template DNA is not of high quality (plasmid)? should I increase the amt of template in the PCR? thank you.
Super fi is very forgiving of poor quality dna and protein contamination even amplifying whole blood templates but it would be better for long amplifications to have good quality dna. I know that the buffer has a constitution that maximises primer binding but I believe that there is a limit to increasing the binding of short primers of low annealing temperatures. I would redesign the primers ( make them longer) to anneal at 64 for long amplification so that the primer has a chance to anneal before the template re anneals with itself. Possibly even try using the highest annealing primer of the set you are using now with the highest annealing reverse primer of the 2 primer sets if this is possible but I really think that 54 is much too low annealing for any primer but especially for long pcr and it must be made longer.
If the quality of the dna is low it is probably better to use less not more dna. PCR relies on copy number not the total amount of dna and plasmid dna is very small so you only need 0.1-10ng of plasmid dna. ( but if it is contaminated with bacterial genomic dna you will need more)
Check your primer amounts. With hot start enzymes you should not be getting primer dimer unless your primers are very badly designed and using less primer will help minimise primer dimer.
If both of your primer sets amplify approximately the same amplimer look at all permutations of forward and reverse primers to see which has the least chance of making primer dimer and try all in pcr s if that makes sense with your use of the amplimer
You could try a smaller reaction volume (20 ul), sometimes that helps with larger templates. You could also try adding some magnesium. The smear does look promising, what are you hoping to do with the amplified DNA?
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