Insert phosphorylation or digestion - which should be done first? - (Apr/07/2015 )

Hi,

I'm going to perform cloning, where the vector backbone will be amplified using PCR generating 5' blunt and and adding 3' restriction site at 3' end. Please check my procedure: PCR product needs to be digested with both DpnI (to remove template) and my restriction enzyme. Then needs to be dephosphorylated before ligation.
Insert preparation: DNA fragment amplifiaction using PCR and then I think I would need to introduce 5'P for blunt end cloning (using T4 PNK and ATP?). I need to digest the product with my restriction enzyme as well. Which one of these two reactions should I'll be doing first?

Thank you in advance for any tips!

Hi,

I don't think that your plasmid needs to be dephosphorylated because after the enzymatic digestion you will have a 5' blunt end and 3' sticky end

I would personally never do this, but if you are keen to do things this way, some things to look at include:

1. Phosphorylate the PCR primer on the blunt end BEFORE pcr, not after your PCR product is made. You can order the primer with a 5' phosphate or use PNK on the primer.

2. Make sure you have some 5' extra bases outside of the primer restriction enzyme site to allow for cutting.

3. Process:  PNK primer, purify primer (or order 5' phosphate on the primer); pcr; pcr purification (do not skip this!); RE digestion; heat kill enzyme or purify RE reaction product; ligate.

You will be happier to choose a RE which can be heat kille

Thank you, I think that is a good point.

What is the best way to purify so short oligo (21 nt)? Today I tried to use columns (GeneClean) but the efficiency was very low. I was thinking about PAGE. Is that a good idea?

I would ethanol precipitate it.