Odd point mutations - (Aug/13/2009 )
teresa on Feb 3 2010, 04:08 AM said:
Hi,
I've been having the same problem: sequencing of my plasmid constructs show several abnormal point mutations (PCR: Pfu Ultra II Fusion HS DNA Polymerase; cells: E.coli DH5alpha).
Did you manage to find out the reason for the mutations?
many thanks,
Teresa
I've been having the same problem: sequencing of my plasmid constructs show several abnormal point mutations (PCR: Pfu Ultra II Fusion HS DNA Polymerase; cells: E.coli DH5alpha).
Did you manage to find out the reason for the mutations?
many thanks,
Teresa
I do not know exactly what was happening. I had the problem with both Ultra II, which had been in the freezer a couple years, and Phusion, which had been in the freezer a couple months. We ordered new aliquots of both, I purified fresh gDNA for template, and I haven't had that problem since.
-fishdoc-
I'd suspect the old template -- perhaps some form of acid hydrolysis or depurination occurred due to prolonged storage in unbuffered water?
-HomeBrew-
HomeBrew on Feb 3 2010, 09:46 AM said:
I'd suspect the old template -- perhaps some form of acid hydrolysis or depurination occurred due to prolonged storage in unbuffered water?
That was my thinking as well. When I made the new preps, I resuspended in tris rather than water. A colleague had just started using the enzymes for some cloning and had much less of a problem with mutations than I did. I think she had 1 point mutation in a few sequencing reactions, whereas I was getting up to 4 or 6. Have done a lot of cloning since, with both Phusion and Ultra II, and haven't had any issue with the new template.
-fishdoc-
I have had a similar problem. I was using gDNA as the template for PCR and looking for a 6.5kb amplicon. At first everything went as planned but over time all downstream experiments went wrong giving me zero clones (I was doing this for a couple vectors). My solution was growing fresh cells for mini-prep.
-georgiadave-