problems in cloning

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problems in cloning

Post by sem.meln » Tue Mar 27, 2007 1:45 pm

Hi, may be someone could help!
I am trying to clone a PCR product cut from both sides with EcorI and NheI (NEB both) to the vector cut with the same enzymes. I did the sequencing of the insert (it was OK), the cutting proceeded for 16 hours (I took 3 mg of each one), cut products I run on gel and extract it from it and did the ligation with T4 ligase of Roshe (ON). The transformation was to DH5alpha by heatshock. The results I get were: no growth in vector only, 5 colonies in vector plus insert. I did the PCR to the insert and did not get any product.
May be someone has any idea? Thank you for your time and consideration.

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Post by SororSaudade » Wed Mar 28, 2007 9:50 pm

how did you cut the vector?

what controls did you use in the final PCR screen? (I mean... did you use a positive control?)

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Post by blcr11 » Fri Mar 30, 2007 3:23 pm

I'm a little puzzled. How were you able to confirm the sequence of the insert if the pcr cloning apparently failed? You sequenced the pcr product itself? or you mean that the target gene was sequenced in situ. Or maybe you're trying to subclone from a pre-existing clone to put a gene or gene fragment into an expression clone of some sort.

If you really used 3 milligrams of DNA in your double-digests, you used way, way too much, but maybe you meant 3 micrograms not milligrams? My first guess is that one of the enyzmes didn't cut so well. Just checking the compatability of EcoRI and NheI suggests that, unless you were very unlucky in your choice of buffers, the two are reasonably compatible, and it should have worked. But perhaps one of your enzymes is very old (or maybe one of you labmates accidently forgot to put the tube back in the freezer and rather than fess up, he/she just snuck it back in the tray next day and didn't tell anyone) or something like that and could stand replacing. My second guess is that the insert is unstable for some reason and is getting deleted from the plasmid. 5 putative positives against a background of zero--if it really is zero--is OK and I would look at all five to see if at least one of them has an insert, either by pcr or by double-digests of miniprep DNA. In the meantime, I would just try it again using microgram amounts of DNA. You don't need to use more than 10-50 nanograms of ligation product in your transformations, either. There is a tendency for people to throw in massive amounts of product, believing that more is better, when at best it doesn't help, and at worst is inhibitory.

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