Cloning Problems - #@%$&*^) (Jan/29/2004 )

We are having some problems. We are trying to clone fragments from 900 to 2000 bp. Our strategy is to amplify the fragments by PCR amplification (PFU Turbo) and to blunt the vectors in which we will insert the fragments. The following is our procedure:
1) digest vectors
2) run on gel and gel purify
3) blunt vectors w/ T4 DNA Pol (this should blunt both 3' and 5' protruding ends if I am not wrong)
4) purify again
5) dephosphorylate vectors w/ Shrimp Alkaline Phosphatase
6) run 1 ul of fragments and vectors on gel to get an idea of amounts to add for ligation
7) ligate w/ T4 DNA ligase (Rapid DNA Ligation, Roche)
8) transform bacteria and plate

On diffent occasions we have gotten different results.
Last time no bacteria grew. This time we have a lot more bacteria on the control plate (ligation of dephosphorylated vector alone) than on the other plates (ligation of vector and insert).

Some things that we think may have gone wrong are the following:
- When we plate the bacteria we use a glass spreader which we clean by putting it over a flame. Potentially, we may be spreading some plates when the spreader is still too hot? we press it up against the lid to make sure it is not too hot but are not sure if this is good enough.
- We may be adding too much DNA to the ligation reaction. The protocol says to add up to 200 ngs. Does this make a huge difference?
- We were successful in cloning into one vector the same time that we were unsuccessful with another. This would seem to indicate that something is going wrong with the ligation conditions. We have tried to mantain a 3:1 ration between insert and vector for blunt end ligations as is suggested in the protocol. Do you think it would be to increase the amount of insert?

Thank you for reading this whole message. Any advice would be greatly appreciated.

You didn't mention how you prepare your PCR product. It essential that the termini of the PCR fragment or the vector have 5' phosphate groups. Since your vector is dephosphorylated to prevent self-ligation, either phosphorylated primers must be used for the amplification or the PCR product must be phosphorylated. Since phosphorylation of PCR products by T4 polynucleotide kinase is inefficient, primers should be phosphorylated prior to PCR reaction.

For detail, have a look at this link:
Cloning of PCR products with phosphorylated primers
http://kuchem.kyoto-u.ac.jp/seika/shiraish...products02.html

Hi,Kawaka

Thanks for this information. I thought the primers commercially customed are 5 end phosphorylated already. That is not true. I will keep it in mind.

Usually they are.

I think that your problem lies in 2 parts.

First is the CIP/AP. If not done properly, it won't work like you want it too.... ie. no clones or too many!

When you get plenty of clones in the negative, it shows that you are not doing the CIP correctly. Either there is too much DNA in the reaction or it is not diluted enough. CIP/AP works best at lower DNA conc in the rxn mix.
When you get nothing at all... then you are probably incubating for too long. My experience is that I incubate for about 1-2 hours. If you are using the NEB system, remember that the CIP works at 50% activity in all the 4 buffers (1,2,3, & 4).

The next probelm could be your ligation. Generally blunt end cloning is not easy, especially if both ends are blunt. Best done at 16oC but there is a lot of optimization to do. Do try to add more ligase inthe mix as well... sometimes this helps.

Let us know what works... we are all learning everyday too.

Cheers!

Hi!

I had some problems with blunt end ligation as well.... then I changed from CIP to Shrimp AP, which is completely and unreversably inactivated by heat, and had generally good results ever since...

Maybe it's worth a try for you, too

Mike

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