Ligation of half blunt PCR fragments - (Feb/21/2007 )
Hello,
I am about to start some (tricky) cloning, this is what I had in mind:
Doing a PCR fragment with a cutting site for a restriction enzyme in one end.
Doing a second PCR fragment with a cutting site for a restriction enzyme in one end.
Cut with the enzymes. This should then give me 2 PCR fragments with one blunt end and one sticky end.
Ligate the Blunt ends.
Ligate the whole fragment into a vector with sticky ends.
Is this possible? I've read that I need to do phosphorylation of my primers for this to work. How do I do that? Can I use the T4 Polynucleotide Kinase from NEB? Anything else I should think of?
Many thanks
which enzyme are you planning to use for PCR reaction?
As far as I understand, in your strategy, your restriction enzymes leave sticky ends and you think that your PCR product will normally have blunt ends. I am sorry but this may not be the case. Taq polymerase for example adds extra A's to the ends so the product is sticky ended. you should check the property of your polymerase you are planning to use for your PCR.
good luck, nice strategy though!
I am about to start some (tricky) cloning, this is what I had in mind:
Doing a PCR fragment with a cutting site for a restriction enzyme in one end.
Doing a second PCR fragment with a cutting site for a restriction enzyme in one end.
Cut with the enzymes. This should then give me 2 PCR fragments with one blunt end and one sticky end.
Ligate the Blunt ends.
Ligate the whole fragment into a vector with sticky ends.
Is this possible? I've read that I need to do phosphorylation of my primers for this to work. How do I do that? Can I use the T4 Polynucleotide Kinase from NEB? Anything else I should think of?
Many thanks
if you use a proof reading enzyme (which you should to begin with), the problem of A overhangs goes away.
However, as a matter of principle, blunt end cloning is difficult. But doable. It is not the easiest of things to do.
As you have already noted, to ligate blunt ends produced by PCR your oligoes need to be 5' phosphorylated. 5' phosphorylation can be purchase as part of your oligo, ie you click the option "5 phosphorylation" and pay some extra money. Alternative, as you have already note, you can use PNK (polynucleotide kinase).
So far so good.
But I would like to adment one thing.
I would first do the ligation and only later followed by the restriction digest.
ie
1- I PNK my PCR products. Then ligate with T4 ligase. The ligation reaction will produce concetameres.
2-Next clean up the ligation mix by phenol/chloroform.
3-Followed by restriction digest using enzyme A and B, that would produce the desired sticky ends.
4- Gel purify (if this is possible) and excise the ligated product of correct size.
5- Ligate into vector.
This assume that the restriction enzymes used do not cut anywhere else in either PCR products
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The reason for reordering the ligation and restriction digest is the none specificity of the ligation reaction, (which actually favours the ligation of sticky ends).
if the PCR products where first cut, the sticky ends produced will ligate during the subsequent ligation reaction. Remember, stick ends are naturally 5' phosphorylated. The ligation reaction then forms concatemers of your 2 PCR products, which you will have to later recut with restriction enzymes. Thus the first initial digest fails to serve any purpose. And can be ommited.
So ligate first (the PNK can be added directly into the ligation mix), clean/remove ligase, and then restiction digest.
Other things you could do.
You could ligate by PCR. If you engineered your PCR products so that they share a small region of homology (3'end of Product1 and 5'end of Product2), you can then anneal them together and ligate by PCR.
You need to do 2 asymetrical PCR reactions (where one primer is more abundant then another) to make 5' ssDNA of Product1 and 3'ssDNA of Product2. Once obtained, ,mix the two products together in equal quantities, add appropriate primers and fresh PCR mix and away it goes.
Ligation by PCR assumes that your PCR product's total size is around 5kb and below.
Your strategy should work quite well. You can improve it by putting a sticky end cutter in the middle instead of the blunt end, but the blunt end should work fine in this case. If you do choose to change to a sticky end ensure the sticky ends are not compatible with one another. If you do stay with the blunt end, you will need to phosphorylate it. A neat little trick for those blunt end users is to design a blunt cutting enzyme on the end of your oligo and cut the PCR product with the enzyme to create a 5` phosphorylated end.
Good luck, Rob
I kinda think that this sounds like a huge pain for a straightforward cloning... lots of possible problems etc, is there a reason why you cant T-A clone then cut out and ligate? You can still add your own restriction sites to the primer if you need to maintain reading frame or something, but I think you will potentially save alot of time and frustration if you T-A clone first...