Hi,
I'm having an issue with cloning the full length of my PCR product into my vector. What I have done:
1.) PCR to amplify my insert where primers add 5' NheI and 3' BamHI sites (with 6 extra bases at each end to allow restriction enzymes to bind). I checked the size of this PCR product on an agarose gel and it has consistently appeared to be the correct size at 750 bp. There are no secondary products being formed that I can see on the gel.
2.) I used a PCR cleanup kit to clean the product and digested the product with NheI and BamHI, then used a cleanup kit again to clean the digested product.
3.) I digested my backbone plasmid with NheI and BamHI, then CIP-treated and ran this on a gel and excised the vector fragment and performed a gel cleanup.
4.) Standard ligation overnight at 16C with insert:vector molarity ratio at 3:1
5.) Transformation via electroporation into either DH5a or MC4100 E coli and plated on LB+antibiotic plates
6.) Colony PCR showed most of the colonies have the correct 750bp insert but there are colonies that do not show anything being amplified
7.) Sequencing confirms this...some of the transformants I sequenced have inserts that vary between ~15bp to ~80bp in length. These show the NheI site and the correct beginning sequence but then the insert is truncated and the last 4 bases of the insert along with the BamHI site appear in the sequence.
I have never experienced this issue before so any help would be appreciated. Thanks!
BAMHI shows star activity in glycerol at greater than 5%. Could this result be due to proper cutting at the ends of the sequence but excising out a fragment by star activity within the fragment then re ligation of the BAMhi end and the NHE end which have inserted into the vector?
Hi there,
I suggest you to run your digested insert on gel to compare its profile with the one you have prior digestion. Are you sure there is no BamHI site in your sequence of interest?
It could be that your inserted DNA (or parts of it) is not tolerated by the bacteria and they chop it up hence you get bits of fragment. This could be due to a number of things but most common are tandem repeat sequences, the bacterial system will try an excise these sequences and leave fragments of DNA. Other potential problems are the presence of cryptic promoters that allow for expression of toxic peptides. You may get different sized colonies on your plates or positive colonies take a lot longer to grow out. If it is a major problem try different strains of bacteria, SURE cells tolerate tandem repeats.
I would recommend running the whole PCR product on a gel, cut the right band, and purify your insert from there. Although you don't see them on a gel you may have small unspecific/truncated amplicons that ligate somehow more efficiently into your vector backbone. Most importantly, I would screen a few colonies by NheI/BamHI restriction (I like it better than colony PCR) for correct fragment size and go ahead with one positive clone. In the end of the day all you need is one. Hope this helps!
There is no way DH5alpha would have so many transformants with a small fragment like 15bp. This sounds more like a phenomenon of Sanger sequencing than a problem with cloning...
If you found some colonies with positive full length insert then you are fine!
Hi Parisa, I'm wondering if you have solved the problem?
I'm experiencing somewhat similar trouble, I need to insert a 4.7 kb fragment into the SmaI site of a vector using blunt ligation, and I need it to be inserted in the forward direction. However every time I check the recombinant plasmids using restriction digestion, I got either inverse insertion or strange band (likely from truncated insertion, forward or inverse, ranged from 2-3 kb).
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