Domain-Exchange via Blunt End Cloning of 2 PCR-Products - (Jul/26/2008 )

Hello everybody,

I want to exchange the domains of 2 proteins (A and B ) and I thought it would be the easiest to do it via blunt ends. They should fit perfectly and I don´t want additional nts in my protein oder missing ones.
Now I made a PCR of the whole Vector of Protein A without the Domain and an other PCR with my desired Domain from Protein B.
I used a Velocitiy Poly which should make blunt ends without A-T overlaps.
Then I dephosphorylated my PCR-Product of Protein A, purified both PCR-products and I ligated them over night at 16°C (T4-Ligase/ 50% PEG4000 added)) in different Vector : Domain Ratios but none of the 6 transformations worked.

When I dephosphorylated my PCR-Product I got a little bit confused. Do I really have to dephosphorylate it? Normally you dephosphorylate the Vector when you digested it and not the PCR-Product.
Is it possible that I have to phosphorylate one of my PCR-Products or that I have to use phosphorylated Primers?
Has anyone ever tried of you tried to ligate 2 blunt-end PCR-products?

I hope you understood my problem and someone can give me sone hints. Normally I´m doing more biochemistry and I´am really not into cloning:-)

Thanks,
Pius-V

-Pius_V-

QUOTE (Pius_V @ Jul 26 2008, 02:38 AM)

Dephosphorylation of digested plasmid is used to prevent self-ligation as the ligase will need the phosphate to work. In your case, I'd buy phosphorylated primers for the insert (and not for the vector-PCR), do PCR and ligate. As your first PCR is not phosphorylated, it should not circularize with itself, and as the second one does not contain an origin of replication it wil not yield viable colonies on selection with an antibiotic. Or you can phosphorylate your insert with T4-kinase and then proceed to ligation (has worked for me too).

-vairus-

or put it another way, by default all primers you order are desphosphorylated. You either have to pay extra to purchase 5' phosphorylated primers or us PNK (polynucleotide kinase) to phosphorylate your PCR amplified insert.

I have conducted BamHI-insertA-blunt End, blunt end-insertB-PstI, and vector BamHI/PstI ligation before. That ligation was doable.

However I have never tried a double insert-double blunt end ligation before and would not recommend it. Double blunt end ligations are difficult and a double insert-double blunt end ligation would be more so.

-perneseblue-

Hi,

thanks for the answers. But I have one Problem with the 5`phosphorylated Primers. Why do I really need them? When I´am doing a normal ligation with a cutted , dephosphorylated vector and a PCR-product it works with ordenary primers. But the T4-Ligase needs always a 5`-P. So there has to be always one...?!

-Pius_V-

when you cut the PCR amplified DNA, you form a 5' phosphorylated end.

example

5- ApApCpCpTpTpGpGpApTpGpCpTpApGpCpT -3'

Cut by restriction enzyme

5- pTpGpGpApTpGpCpTpApGpCpT -3'

If you had directly used a PCR amplified product, its 5' end is not phosphorylated

-perneseblue-