There's a clone in pBAD18cm vector (6800bp) which was subjected to point mutation through PCR-based technique. After PCR and DpnI digestion an extra band of approx 1500bp was observed in cloned plasmid sample. Subsequently, after transformation, the extra band size increased to 3000bp (approx) along with expected band of 6800bp.
A control was set up with only pBAD vector devoid of clone with mutant primers, an extra band of same size (1500bp) was still there after DpnI digestion with no other band. But there was no any colony after transformation with the same control.
What could be the possible explanation for this?
This suggests to me that your vector is not what you think it is, in fact it would have be be either the wrong vector you have or somehow the version you have has a mutation it it that cuts with DpnI. Have you sequenced your vector, or at the least cut out the extra band and sequences that? That might tell you where its from in the vector and what its ends are
Hi there..
Thanks for your response. The vector seems okay as I have prepared several mutants using the same cloned vector but only one clone displayed such observation. The vector with clone has been sequenced previously and was stored in -80 in XL1 blue cells.
DpnI only cut the methylated DNA (your template DNA) not PCR products. Full-DpnI digested pBAD should have the largest fragment of around about 900bp ( you can do a DpnI digestion analysis of your construct to check the digestion profile). However due to your DpnI-digestion condition is not optimized, it is possible that some partial digestion product of 1500bp remained.
Usually after DpnI digestion, you just check the presence of full-length PCR product (vector plus cloned gene), don't bother too much about the digested fragments. Your PCR product without methylation shouldn't be digested by DpnI and it should shown in full-length. If no full-length DNA fragment after DpnI digestion, this mean your mutagenesis PCR amplification did not work and you need to modifying your PCR cycles, some time even don't bother do the transfomation. If there was the full-length products but no colony after transformation, and if that is the case you need to optimize your transformation protocol.
There is optimised mutagenesis protocol (http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91) more efficient than the Quickchange protocol, you can follow the modified protocol if you experienced low PCR amplification efficiency.
Thanks Huanting Liu. Your explanation is helpful to me. I do get the vector+clone sequence after DpnI digestion and one of the sample showed this extra band similar to pBAD control while others did not. Anyway, I screened some more colonies and found other fine and reconfirmed by double digestion. The sequencing results are pending for further confirmation.
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