sequencing - which primer should I trust? (Dec/10/2006 )
Hi everyone,
I met a problem with a sequencing, actually I made a point mutation and when I look at the sequencing I got the right mutation only on the forward primer so I could trust this primer since the chromatogram seems not too bad at the mutation's place but I also got an addition of 4 nucleotides which is not present in the reward primer. I could sequence again but it takes 3 days before getting the results 
and I wanted to purify the protein right now. What would you do?
I would wait for the resequencing results.
How many plasmids isolates are you sequencing? Do you have more then one isolate of this plasmid type? I would actually sequence at least 3 difference isolates of this plasmid as a precaution that one or more plasmids contains a sequence error.
However, if you don't mind burning time (if there is an error), then go ahead and do the protein induction.
How many plasmids isolates are you sequencing? Do you have more then one isolate of this plasmid type? I would actually sequence at least 3 difference isolates of this plasmid as a precaution that one or more plasmids contains a sequence error.
However, if you don't mind burning time (if there is an error), then go ahead and do the protein induction.
Thanks for your reply. I have used only one isolate plasmid so maybe I should purify an other isolate plasmid which might not contain the addition of nucleotides. Anyway I'm gonna wait for the second sequencing.
C U.
I would take 2 or 3 colonies to and do sequencing per duplicate. And I would wait for resequencing too. If not, there is a risk you’ll waste time and reactives!!!
Yes, I agree with others. It's better for you to sequence a few more colonies (another one or two), rather than wasting your time for the protein work if it is the wrong plasmid.