Different colonies sequence - (Sep/26/2009 )
Hi,
I transformed some DH5a cells with a certain plasmid that a senior had left behind. I obtained many colonies as I should. I miniprepped and sequenced from one of the colonies and found two unwanted mutations (in the corresponding protein sequence). I have been advised to pick another colony and re-sequence. My question is can different colonies have different sequences. I think the answer is a 'theoritical yes' as different colonies arise from different precursor cells, in that case - is different sequences common amongst different colonies that were raised from cells transformed with the same plasmid?
Thanks!
arnabde2000 on Sep 26 2009, 05:00 PM said:
What is the source of the plasmid which you used to transform your cells? If it is insertion of PCR product into a vector than every colony has a different copy of DNA and answer is yes. But if it is a clone-derived plasmid, every transformed cell/colony will be the same.
It is possible, sometimes the DNA can be mutated during the replication of the plasmid.
Hi gleb, it is a clone derived plasmid. Could you please explain why a clone derived plasmid should lead to every clone being the same and why would it be different for a PCR product. Thanks!
Hi bob, is it common to see this difference during transformation or is it just a theoritical mishap?
arnabde2000 on Sep 27 2009, 11:28 PM said:
Because Taq has a natural accuracy about 1 mismatch on every 1000 b.p. since bacterial replication has 1/10^6.
That means long PCR product will contain mutations with a much higher probability than cloned product. And if you want to clone PCR product for purposes of engeneering or something you have to screen appropriate clones with no mutations (or insignificant one).
arnabde2000 on Sep 27 2009, 11:28 PM said:
Hello arnabde
Did you check the chromatograms yourself or you got the sequences from some company?
Was your sequence a result of a consensus of two or more separate sequencing reactions (even from the same colony)?