at the moment I am doing site directed mutagenesis using phusion enzyme. the procedure worked every efficiently but now while I was screening successful colonies by sequencing I was observing a number of errors which I suspect that were introduced during the cycle sequencing procedure. I am thinking this way because from the same miniprep, I sequenced twice using two primers creating an overlap of about 100bp or so. For example one sequence from primer 1 had a G at particular position while when using primer 2 instead of a G I found an A and this is supposed to be the same as the miniprep is coming from the same clone. I noticed that these errors are either a G replaced by an A or a C replaced by T. Did anyone encountered something similar please?
thanks
-dnalab-
Sequencing results are only accurate for about 400 bp with F primer and 300 bp with reverse primers. Did you amplify the sequence or do a miniprep. Taq and pfu have error rates for synthesis, taq more that Pfu, so if you amplified your sequence, there will be some errors in the sequence.
--SR
QUOTE (dnalab @ Sep 7 2008, 05:39 AM)
at the moment I am doing site directed mutagenesis using phusion enzyme. the procedure worked every efficiently but now while I was screening successful colonies by sequencing I was observing a number of errors which I suspect that were introduced during the cycle sequencing procedure. I am thinking this way because from the same miniprep, I sequenced twice using two primers creating an overlap of about 100bp or so. For example one sequence from primer 1 had a G at particular position while when using primer 2 instead of a G I found an A and this is supposed to be the same as the miniprep is coming from the same clone. I noticed that these errors are either a G replaced by an A or a C replaced by T. Did anyone encountered something similar please?
thanks
-DazedNConfused-
QUOTE (DazedNConfused @ Sep 8 2008, 02:55 PM)
Sequencing results are only accurate for about 400 bp with F primer and 300 bp with reverse primers. Did you amplify the sequence or do a miniprep. Taq and pfu have error rates for synthesis, taq more that Pfu, so if you amplified your sequence, there will be some errors in the sequence.
--SR
QUOTE (dnalab @ Sep 7 2008, 05:39 AM)
at the moment I am doing site directed mutagenesis using phusion enzyme. the procedure worked every efficiently but now while I was screening successful colonies by sequencing I was observing a number of errors which I suspect that were introduced during the cycle sequencing procedure. I am thinking this way because from the same miniprep, I sequenced twice using two primers creating an overlap of about 100bp or so. For example one sequence from primer 1 had a G at particular position while when using primer 2 instead of a G I found an A and this is supposed to be the same as the miniprep is coming from the same clone. I noticed that these errors are either a G replaced by an A or a C replaced by T. Did anyone encountered something similar please?
thanks
thanks for reply
-dnalab-
It would be very surprising to truly see different sequencing results with two different primers from the same miniprep DNA. I'd suggest looking at the electropherogram rather than the called bases to see what is really happening. With modern sequencers and good reactions you now get 800-1000 bp of good sequence. You should easily be able to sequence between a forward and reverse primer located 1000 bp apart, but it may require some intelligent analysis of the electropherogram.
-phage434-
As Phage suggests, i would take a closer look at the electropherograms to confirm that the sequences you are refering to are correct.
-killerkoz17-
