Blunt ended ligation - (Dec/21/2005 )

Hey,

I have to insert a 240 bp and also a 2.7kb insert into a 11 kb vector using a PmeI site. I have never perfromed a blunt ended ligation having always prefered the advantages associated with sticky end ligations. Would the following methodology work?

Design both forward and reverse primers with phosphorylated 5' ends.
Amplify inserts with proofreading polymerase (Pfx) to obtain blunt ended product.
Gel purify the PCR product
Digest vector with PmeI (blunt ended restriction site)
Add Antartic Phosphatase to the restriction digest after O/N Digestion
Run out vector on gel and excise and purify product
Ligate the vector to the insert in a 1:3 molar ratio.

I am just checking this is OK as I obtained no colonies last time although I heat inactivated the phosphatase (possibly leaving it still active?) rather than gel purify last time.

Thanks

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Hey everyone,

I really need to get this to work and had tried the protocol above with one difference and it did not work. The difference was I gel purified my product and then treated with the phosphatase before heat inactivation at 65oC for 20 minutes. I got no colonies from the resulting transformation in vector + insert or digested vector alone reactions. Does anyone have any suggestions. My sticky ended ligations work but this is my first attempt at blunt ended ligation.

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did you do a positive control? your strategy looks OK to me, but something must be off...how are your comp cells?

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How are you doing the ligations? I'd recommend the NEB quick ligase kit, which has a PEG containing buffer, accelerating the ligation of blunt ended inserts.

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I agree, try adding some PEG to the ligation reaction. You may also want to try other vector:insert ratios.... Apart from that though, your strategy sounds pretty good. Blunt ended ligations are often difficult, and I had success mainly when ligating short blunt ended fragments into small vectors. But as they say, you only need one!! As amikins said, I think having excellent compentent cells is crucial. What compentent cells are you using and how are you generating them? Are you getting much background ligation of the vector circularising?

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Thanks for the advice,

I have performed the ligations twice and I have had nothing on all plates. All plates being vector plus insert (different ratios from 1:3 to 1:10 used) and a negative control was vector only (which was digested and SAP treated vector following by gel purification). I did not use a positive control. I am using chemically competent Stbl3 cells as these were recommended for use with unstable lentiviral vectors and I had used a vial from this pack of competent cells to maxiprep the lentiviral vector. After the first attempt I was dubious about the ligase so used a T4 ligase from a TOPO TA kit but as already mentioned the second round of ligations have also resulted in no colonies in negative plate and vecotr + insert plates. I have included an attachment which shows the PmeI digest of the vector and also a gel of the two fragments I am trying ligate into the vector following gel purification.

Kind regards,

Pete

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I have used the T4 ligase from invitrogen.

Cheers,

Pete

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Hi,
First, you should remove any traces of phosphatase (Maniatis recommend phenol/chlor). At least, your fragment must be gel-purified.
Second, for ligation you need concentrated ligase (NEB has it)
Third, the fragment need to be ligated overnight at 16C.
Good luck!

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