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Problem cloning wild type E. coli gene... Possible recombination event in DH5alp


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#1 Tscott760

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Posted 17 May 2011 - 04:15 PM

Hey everyone!

So I am trying try clone a gene of interest (around 480bp's) that I have PCR amplified from purified E. coli(DH5aplha) genomic DNA. After PCR I get a nice band right where it should be so I know my insert is good. Primers from Sigma contain KpnI and ClaI RE sites with a few extra bases at each end for efficient digest.. I purified my vector from the DM1 cell line so that there should be no digest problems due to methylation. I digested each individually, heat deactivated, dephosphorylated, and spin column purified my vector, so that should be good too.

Ok so here's the problem... My cut and purified vector runs at 5kb prior to ligation, but then after I ligate my insert and transform it back into DH5aplha cells it seems that I am missing a 1 kbp region of the vector which includes the GST tag/promoter and the multiple cloning site! After weeks of retrying and starting from scratch, I always end up with the same 1 kbp fragment missing for all 10 or so colonies per ligation... I have sequenced the entire vector before and after ligation, verifying this region has been removed and I have checked to make sure there were no extra cut sites anywhere...

Here are the regions of the original vector sequence that seem to ligate to each other only when trying to clone my insert: (79)5' ...TAAATCACTGCATAATTCGTGTCGCTCAAGGCGCA...3'(113)..... (1160)5'...AGTGTATAATTCTTGAAGACGAAA...3'(1183)

This is the sequence I get after ligation... 5'(79)...TAAATCACTGC*ATAATTC*TTGAAGACGAAA...3'(1183)

I noticed a some sequence homology between the 2 regions of vector coming together. Any chance there may be some sort of recombination event going on in the cells even though DH5alpha cells lack RecA???

Trevor

#2 pDNA

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Posted 18 May 2011 - 12:05 PM

uhh ...that looks like recA independent recombinantion!

You can find interesting details on structural instability of plasmid DNA in that review:
My link

Regards,
p

Hey everyone!

So I am trying try clone a gene of interest (around 480bp's) that I have PCR amplified from purified E. coli(DH5aplha) genomic DNA. After PCR I get a nice band right where it should be so I know my insert is good. Primers from Sigma contain KpnI and ClaI RE sites with a few extra bases at each end for efficient digest.. I purified my vector from the DM1 cell line so that there should be no digest problems due to methylation. I digested each individually, heat deactivated, dephosphorylated, and spin column purified my vector, so that should be good too.

Ok so here's the problem... My cut and purified vector runs at 5kb prior to ligation, but then after I ligate my insert and transform it back into DH5aplha cells it seems that I am missing a 1 kbp region of the vector which includes the GST tag/promoter and the multiple cloning site! After weeks of retrying and starting from scratch, I always end up with the same 1 kbp fragment missing for all 10 or so colonies per ligation... I have sequenced the entire vector before and after ligation, verifying this region has been removed and I have checked to make sure there were no extra cut sites anywhere...

Here are the regions of the original vector sequence that seem to ligate to each other only when trying to clone my insert: (79)5' ...TAAATCACTGCATAATTCGTGTCGCTCAAGGCGCA...3'(113)..... (1160)5'...AGTGTATAATTCTTGAAGACGAAA...3'(1183)

This is the sequence I get after ligation... 5'(79)...TAAATCACTGC*ATAATTC*TTGAAGACGAAA...3'(1183)

I noticed a some sequence homology between the 2 regions of vector coming together. Any chance there may be some sort of recombination event going on in the cells even though DH5alpha cells lack RecA???

Trevor



#3 Tscott760

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Posted 19 May 2011 - 11:52 AM

Thanks for that interesting article, it seems there are other people that have had trouble with RecA-independent recombination events with the pGEX vector series.. As for solutions to this problem, my only ideas are to either do site-directed mutagenesis on one the homologous ATAATTC sequences since they don't appear to be in critical regions of the vector... I would prefer not to do this in order to minimize the chance of having taq-induced mutations elsewhere in the vector..

Does anyone know of an E. coli strain that is deficient for any other RecA-independent recombination genes??

#4 pDNA

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Posted 19 May 2011 - 12:19 PM

for cloning direct repeats or complicated structures you can try Stbl or SURE cells

short info SURE-cells:
"The SURE (Stop Unwanted Rearrangement Events) strain* was engineered to allow the cloning of certain DNA segments that are
“unclonable” in conventional E. coli strains. The SURE strain lacks components of the pathways that catalyze the rearrangement
and deletion of nonstandard secondary and tertiary structures, including cruciforms (caused by inverted repeats) and Z-DNA, that
occur frequently in eukaryotic DNA and that impede the cloning of the eukaryotic DNA in conventional strains. SURE cells are
restriction minus (McrA-, McrCB-, McrF-, Mrr-, HsdR-) endonuclease (endA) deficient, and recombination (recB recJ) deficient.
The lacIqZΔM15 gene, on the F´ episome, allows blue-white screening."


shor info Stbl2:
"MAX Efficiency® Stbl2™ Competent Cells are high-efficiency chemically competent cells specifically designed for cloning unstable inserts. In addition to recA1, a unique set of genetic markers allow for stable cloning of direct repeat and retroviral sequences (1) and tandem array genes (2)."

there are others as well ...but i do not remember their name now, sorry!

Regards,
p

#5 Tscott760

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Posted 17 June 2011 - 03:14 PM

Ok so I tried cloning this gene into a different vector using different restriction sites and still got similar results that seem to cut out ~ 1Kb. I cloned a mouse gene into the same vector sample I used for the problematic gene and that worked fine first try... I'm starting to think maybe my transformation procedure could be affecting/facilitating the recombination event, if that's what is really going on.. I transform on ice for about 45-60 min, then add pre-warmed SOC for another 30-60, then plate. I get tons of colonies but every one I pick (up to 20 at a time) seems to be missing that 1kb... Any chance this may be too long on ice? Anybody else notice anything out of the ordinary when varying transformation protocols? I normally don't however this particular gene is becoming quite a menace!

T




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