pcr mutagenesis - (Jun/17/2007 )
I am trying to add a 15nt oligonucleotide to the 5´ end and a 6 nt oligonucleotide to the 3´end of a sequence which places in a plasmid by PCR. My forward primer is 34nt and it has 15nt overhang and reverse one is 27nt and it has 6nt overhang.Their Tm are >100 and 68 respectively but,i could not see any band at 65 temprature and lower tempratures up to 53. At 53 as annealing temprature i saw alot of bands .I have tried gradient temprature PCR and different cosentration of Mg but it has not worked.Any hints?
soly, if i understand this correctly, your sense primer (5' to 3') has 15nt overlap, a mutated region (how many nt?), and then 6nt overlap with the sequence you wish to mutagenize on your plasmid? if this is correct, i would get new primers. i would not use such a short overlap on your 3' end and for the new primers i would extend it to at least 12-20nt. the longer the region you wish to mutagenize, the more the overlapping sequence you are going to need. and for the reverse primer, i would make sure it is exactly antisense to your mutagenic sense primer. i hope this helps,
mateo
dear mateo
I think there is a misunderstanding , there is not any overlap , we are trying to add a 15nt to the 5´end of our sequence so we added this 15nt to the 5´end of my forward primer which is 35nt . as a result there is only 20nt base pairing to the sequence and 15nt of primer is overhang.in addition to we are adding a 6nt to the 3´end of our sequence too so we have a reverse primer which is 27nt and 21nt can match to the sequence but 6nt at 5´end of the primer is overhang I hope I could explain it clrearly. I d appreciate it if you can help me
I think there is a misunderstanding , there is not any overlap , we are trying to add a 15nt to the 5´end of our sequence so we added this 15nt to the 5´end of my forward primer which is 35nt . as a result there is only 20nt base pairing to the sequence and 15nt of primer is overhang.in addition to we are adding a 6nt to the 3´end of our sequence too so we have a reverse primer which is 27nt and 21nt can match to the sequence but 6nt at 5´end of the primer is overhang I hope I could explain it clrearly. I d appreciate it if you can help me
sorry about the confusion on my part. one question I have is how you calculated the Tm for the primers. you say that the first one is > 100C, which sounds very high to me especially for only 20nt annealing. your Tm values should be calculated only for the annealing region of your primers and not the adaptors. so if your estimate is off, i would retry the pcr using the correct annealing temperature.
two other important questions are the source of DNA you are using as a template (genomic, plasmid, bac, etc) and how long the final amplicon will be?
You are right, my annealing temperature is high but it was company s suggestion and they did not consider the part which doesnot anneal so we used temperature gradient. At first it used to amplify at 48 and 50 but after some time we did temperature gradient again and this time it could amplify at 61 and lower too but in both situations it has lots of unspecific bands.as to the template ,it is a cDNA cloned in blue script plasmid and the final amplicon is 5 min.
thank you for your help