I need help --- ligation troubleshooting - (Mar/25/2006 )

I have trouble in cloning a 1.4kb PCR product into pET-28b vector (5.3kb).

I double digested both PCR product and pET-28b vector with Hind III and Nde I. Put them in a ligation (insert 6uL, vector 2uL, NEB T4 ligase 0.5uL, 10X ligase buffer 1uL, water 0.5uL). Incubate at room temperature for 16 hours and then directly transform my 10uL ligation into 50 uL TOP10 competent cells. However, after 16 hour incubation at room temperature, I got no colony at all.

I think something in the ligation mixture may inhibit the transformation. Do I need to use the QIAGEN PCR purification kit to purify the ligation mixture before I proceed to the next transformation step? Do you have good suggestions for me. Thanks a lot!

what have you done for positive controls?

your ligation mix shouldn't inhibit the transformation, unless there is something in your vector or insert that inhibit the ligation reaction itself

I think 16 hours at RT is awfully long at that high temperature, but I've never tried it before; anyone else do it that way? I ligate at 16 degrees C O\N, or at RT for 20-30 minutes

I only have a negative control --- linearized vector only (also no colony). 37 might be too high! I might probably try a 16 degree O/N to see what I will get.

I would add a positive control or two, as well, to be sure everything's working properly

this is what I would suggest:

uncut plasmid vector only / transform --> tests competent cells, transformation protocol...plate on media with and without antibiotics; checks also your selection...you can use this to calculate transformation efficiency if you want, as well

when you are running through your protocol, cut your vector with a single-cutter, purify it the same way you do your other fragments, and re-ligate it back together, with no phosphatase step / transform --> this tests your ligation protocol and reaction components, as well as your purification steps

if everything is working properly (lots of colonies on above two controls) and you're still not getting any transformants, I guess next you would look at your RE's and your PCR.

good luck!

I would add less of your ligation product to your Top10 cells. In our experiments, anything more than about 5% volume of ligation mix into the chemically competent (Invitrogen) cells started to become inhibitory. Also, you didn't say what resistance your plasmid has. For anything other than Amp, you'll need to let the cells express the resistance gene for an hour in non-selective broth prior to plating them out on selective plates.

oh, they are Kan resistant and I let them grow for about 2h in SOC before plating them.

Thanks you all for giving me so many suggestions. BTW: Have you ever successfully cloned PCR fragment (with restriction sites) directly into vectors without using TA cloning method? If so, could you tell me what your starting amount (I mean for digestion) of PCR fragment is, 1ug, 2ug, or more?

If you are cutting a PCR fragment and attempting to ligate it with a vector backbone, it is important to purify the DNA prior to RE cutting. Otherwise, the still-active PCR enzymes will extend your cut DNA fragments and destroy the overlapping ends you rely on for the ligation.

fpr that purpose, purify DNA by phenol chlo and etoh precip
that do my first digestion, purify it by phenol chlo and etoh precip second digestion and purify by column. its ok and works better than with gel purifs

Dear friend
First It is documented that Ligase inhibits transformation. Therefore if you have not denatured ligase before using transformation mix for ligation then possible you may end up in a result you have got.
Second, are you sure that your competent cells are good enough to show low frequency of tranformation associated with directional cloning as in your case.
Equaly likely is a possibility that you have lost cohessive ends during DNA purification and hence no ligation, no colony.
!6 hrs at 16C work well at hour hands.
SPandey