5 replies to this topic

[Sharky]

Hey guys,
             So basically, I have sequenced my PCR product and made sure its the same as the wild type gene from the genome. After verifying, I ligated it into the pet151/D-TOPO from Invitrogen, which has a His tag/V5 epitope and TEV protease cleavage site at the N-terminal of the protein. I have then transformed TOP10 cells, screened for positive colonies, and isolated the plasmid of those ones that have the insert in the right orientation. I have saved the positive colony in the -80.
I performed transformation of BL21 and pilot expression has shown that I have no clear overexpression in both soluble or insoluble, I have verified my expression conditions are correct and tried optimisation by inducing and growing at lower temperature, range of IPTG conc (0.5mM-1mM) and nothing. I know my gene is not toxic also.

Is it possible that the insert is not in frame with the plasmid, because of a mutation happened to the DNA after ligation?

[fishdoc]

Sharky, on Mar 25 2010, 09:49 AM, said:

Hey guys,
             So basically, I have sequenced my PCR product and made sure its the same as the wild type gene from the genome. After verifying, I ligated it into the pet151/D-TOPO from Invitrogen, which has a His tag/V5 epitope and TEV protease cleavage site at the N-terminal of the protein. I have then transformed TOP10 cells, screened for positive colonies, and isolated the plasmid of those ones that have the insert in the right orientation. I have saved the positive colony in the -80.
I performed transformation of BL21 and pilot expression has shown that I have no clear overexpression in both soluble or insoluble, I have verified my expression conditions are correct and tried optimisation by inducing and growing at lower temperature, range of IPTG conc (0.5mM-1mM) and nothing. I know my gene is not toxic also.

Is it possible that the insert is not in frame with the plasmid, because of a mutation happened to the DNA after ligation?

You should sequence the plasmid to know if it is correct. Sequencing the DNA used for ligation will only give you the "average" sequence in a mix, and it's possible a piece that had a point mutation was present and inserted into the vector. Once it's in the vector, the sequence (correct or incorrect) should be pretty stable, so always sequence the vector.

[Sharky]

Sent in the plasmid for sequencing! Hope I dont have any mutations:D

[DyDx]

Personally, I never sequence the PCR products, just the resulting plasmid. If you're using something with proofreading, like Pfu, you should almost never have mutations for standard PCR.

[Denny]

I agree with Fishdoc and DyDx, I always sequence the plasmid construct to make sure I know exactly what sequence has been inserted, and that it's in frame among other things. The PCR will be a mixture of sequences and you can't guarantee that the PCR sequence you verified is the same sequence you ligated to the vector.

Edited by Denny, 25 March 2010 - 03:06 PM.

[Judes]

Sharky- To clarify, do you use BL21 or BL21(DE3)? If the former, then I would switch to BL21(DE3). BL21 does not do well with IPTG induction. Hope that helps!