blunt end ligation - Help (Jan/27/2006 )

Hi every one :

Good evening!

Now I encounter a problem about blunt ligation. I konw that blunt ligation efficiency is much lower than cohensive end ligation. My vector is 21kb, and exogenous insert is 4.5kb, I wonder whether it is viable. and any optimization can be done to improve its liagtion efficiency? Any suggestion is appreciated! Many thanks to you!

Cinba

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There is one major optimization you can do...dephosphorylation of the digested vector. I would suggest using Shrimp Alkaline Phophatase (SAP) as it is by far the easiest one to use, in my humble opinion. In case you haven't been exposed to this before, SAP will remove the phosphates at the 5' end of the DNA strands on both ends of the digested vector. This will prevent it from self-ligating to its own 3' OH groups. You can of course use Calf Intestinal Alkaline Phosphatase (CIAP) but to be honest it can be a pain in the buns to inactivate. SAP can be inactivated in 15 minutes at 65oC and is compatible with many restriction buffers.

A second thing would be of course to ensure complete digestion (as complete as possible anyway) by giving the digest reaction a good run time (minimum of an hour, I have even gone overnight or over-weekend but the weekend was just because I wasn't in the lab ).

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I have been testing out NEB's new Antarctic phosphatase...it's not bad. If you follow the instructions it is not 100% but the efficiency is pretty high compared to no-phosphatase control (yeah, I'm a geek, I had to test it; used 3' overhangs)

anyways, it's not terribly expensive if you're not going through too much, it works in any RE buffer (supplemented with it's own buffer too) and it heat-inactivates in 5 minutes

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Hi Captain DNA and Aimikins:

First, Thanks for ur Guides and ur suggestions are much appreciated!

But, besides, Are there any other methods to improve the efficiency Blunt ligation between large fragment?

More suggestion is welcome!

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There was a nice buffer from BRL which was specially made for Blunt end ligation. This buffer contains PEG (polyethylene glycol)which is apparently helping to made the contact between DNA species much closer. I had quite a lot of success with this one when I was doing Viral vectors constructs (Big vectors and inserts).

I took that out of a paper
Abstract

The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of high concentrations of a variety of nonspecific polymers such as polyethylene glycol, Ficoll, bovine plasma albumin, or glycogen. Blunt-end ligation of small self-complementary oligodeoxyribonucleotides is also stimulated. The use of polyethylene glycol 6000 in such systems is characterized in some detail. Conditions are suggested which either greatly increase the rate of formation of and size of linear ligation products or which allow smaller but significant improvements in the amounts of circular ligation products.

Hope it helps if you run a google search you will get plenty hits (use of PEG for blunt end ligations)

Pesji

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Hi pesji:

Thanks for your suggestion!

best regards!

sincerely yours

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If you switch to these buffers, do NOT heat kill the ligase. Heat killing ligase in PEG containing buffers reduces transformation efficiency by 100x or so according to our experiments. This does not happen in normal ligation buffers.

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You're welcome !

pesji

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Hi Phage434:

Thanks for your kindly reminding!

If I want to add PEG into the ligation RXN system. before transforming E.coli, should I purify the ligation product? or I could use it without any treatment ?

Many thanks!

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We just transform into chemically competent (Invitrogen Top10) cells. If you are electroporating, it might be necessary to dialyze to remove salt, but I'd try it without doing anything first. The volume of ligation mix must be < 5% of the cell volume for best efficiency in chemical transformation. If you don't have enough colonies from this you'll have to purify the DNA first.

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