3 replies to this topic

[aea_imp]

Hi,

I wonder if anyone can offer me any advice. I've been trying to clone the promoter region of a mouse gene which I had previously sequenced. I'm using genomic DNA from a C57BL/6 mouse as starting material. I've used the Roche Expand High Fidelity Enzyme to amplify up this product. I have then digested it with 2 restriction enzymes, ligated it into a pGL3-basic vector from Promega, transformed JM109 E. coli and minipreped a number of colonies. I screened the colonies and sent the ones with the correct insert for sequencing. The problem I am having is that the sequences I received back all have errors (up to 4 bp errors per clone, but not all the clones have the same errors) when compared to my original sequence (which I am certain is the correct sequence). All the clones were made using the same PCR reaction product. Are these errors occuring in the restriction digestion, ligation or transformation step? Can anyone suggest anything I could do to stop these errors occuring?

Thanks for any help you are able to provide.

Amy Anderson

[lyrezxl]

Quote

Are these errors occuring in the restriction digestion, ligation or transformation step?

i don't think so. Generally, digestion, ligation and transformation will not cause the mutant.

you don't give enough infor. how many bps of your fragment? how about your PCR? ......

i never used the Roche Expand High Fidelity Enzyme.

and before you sequencing your clone, maybe you'd better check it by some  method first. PCR, RE digestion or something else.

hope it helps!

[aea_imp]

Hi,

Thanks for your reply.

My PCR product is 1086bp long. I follow the protocol sent with the kit (long-range protocol). I clean the PCR product with a Promega kit, digest it with Xho I and Mlu I, clean that reaction and then ligate into the vector.

After I have minipreped the colonies I use a small sample of this in a restriction digest to make sure I'm getting the correct sized insert.

I really can't work out what is going wrong. As I said I have different mutations in colonies which come from the same PCR reaction/ligation reaction.

Amy

[tfitzwater]

PCR errors are typical, even with high fidelity enzymes.  We use three different DNA polymerases for each cloning project: Pfu Turbo, KlenTaq and Pwo.  Pick two colonies from each version and sequence the plasmids.  Hopefully at least one of them will be correct.  If not, we generate a restriction site analysis and figure out how to cut and paste fragments with the correct sequences together.