oligos phosphorylation with PNK - (Feb/11/2008 )

Does anybody know how to know the phosphorylation is successful? Thanks.

-kongkong-

If you radiolabel them, run on gel.
Most oligos are phosphorylated when you purchase them.

-mikew-

Most oligos are definitely NOT phosphorylated when you buy them. Adding a 5' phosphate is an extra cost option.

-phage434-

Hi again,

Phage434 is correct. I was wrong. You need to request
5' phosphorylation. I apologize. I actually tried to anneal and clone
oligos a long, long time ago and it didn't work for exactly this reason.
Have said that, other than radiolabeling I am not sure how to
determine if oligos are actually labeled. Their migration in a high percentage
polyacrylamide gel may be slightly different but this is a long shot.

-mikew-

You dont need to phosphorylate if you digest the linker first right if it has restriction sites at the end with some extra nucleotides? Coz ive been having problems with this, actually been trying to ligate a plasmid for 6 months (on and off). But so has everyone else in this department...

-gorkin-

That's right. Restriction enzymes will leave a 5' phosphate when they cut.

-phage434-

QUOTE (phage434 @ Mar 4 2008, 11:30 AM)

That's right. Restriction enzymes will leave a 5' phosphate when they cut.

So it must be something else thats inhibiting the reaction? Every here is having problems. Maybe its the water since all the reagents we use are brand new?

-gorkin-