Sequencing different colonies - (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, 08:00 PM said:

There probably won't be a difference in the sequence. However, it's possible the mutation occurred after transformation. It's not a good possibility, but it could happen. Sequencing a second (or more) clone will tell you if the plasmid carried the mutations to begin with or if they were introduced in transformation or by the E. coli strain. If possible, sequence the plasmid from the original source, too.

If another clone gives me the same mutation, the it would probably mean that the plasmid carried the mutation to begin with. If the other clone gives me the right sequence without mutation, then it would mean that the mutation occured during transformation. Am I correct? Also would it be more technically correct to say that in the second case, the mutation occured not during the transformation but during the replications and cell divisions after the transformation? I thank you for your help!

arnabde2000 on Sep 26 2009, 10:10 PM said:

That is correct in my estimation. I think it's highly unlikely the mutation occurred during replication in E. coli, but verifying that multiple clones have the same mutation is needed. It could have also been a mistake in the sequencing, so you should check the chromatographs if they're available.

I think fishdoc mentioned this, but if you sequence plasmid from the original miniprep used to transform your cells (if you have any left) and the mutation is there, them there's no sense in sequencing further colonies. On the other hand, if the mutation is not there, you can sequence further colonies, or re-do the transformation.

Also, if you suspect it's a sequence base-call anomaly, sequence from the other direction, or with a different primer such that the area in question is in the strong part of the trace.

It is not a sequence anomaly. I checked the chromatograms and the mutation is REAL.