3 replies to this topic

[xyz74]

I have a question about plasmids. I amplified my cDNA into pOTB7 vector (chloramphenicol resistance). I did the sequencing and got 100% match with my gene of interest.

Then I RE digested this plasmid and pcI-neo (amp resistance) and did ligation etc. When I sequence this new plasmid, I am getting about 10 mutations in each primer that I used.

I am confused because the first plasmid is fine but the second one is full of mutations. Also the RE sites in pcI neo are only 5 bases apart. Does that have anything to do with the mutations?

Thanks.

[Nrelo]

xyz74, on Feb 24 2009, 05:40 PM, said:

I have a question about plasmids. I amplified my cDNA into pOTB7 vector (chloramphenicol resistance). I did the sequencing and got 100% match with my gene of interest.

Then I RE digested this plasmid and pcI-neo (amp resistance) and did ligation etc. When I sequence this new plasmid, I am getting about 10 mutations in each primer that I used.

I am confused because the first plasmid is fine but the second one is full of mutations. Also the RE sites in pcI neo are only 5 bases apart. Does that have anything to do with the mutations?

Thanks.

You got only one construct/clone (I mean the second one)? If not, did other clones give you the same problem?

[xyz74]

I checked only one clone so far, I am thinking of picking more colonies and sequencing them also to see if the problem continues.

[scolix]

I would guess something is wrong in one of the 2 vectors. Better go back to the original plasmid and verify sequence again. Also look at the original sequence data. They can give a better idea.