re-dephosphorylation of vector? - (May/26/2006 )

i cut my vector with stuI and dephosphorylated but i got all self ligated colony

can i dephosphorylate the vector (alreadydephosphorylated) again or i should dephosphorylate new vector


thanks in advance for ur kind reply

Hi. You can do dephosphorylation again.

I've had the problem before and kinda troubleshot it. Sometimes, it's the dephosphorylation inefficiency but most probably it's the undigested plasmids that you should be worried more.

If you digested your plasmid, it's advisable to run on agarose gel and gel isolate the fragment that's cut. That'll ensure that uncut plasmids will not enter the dephosphorylation step. Only then, do dephosphorylation.

after cut with enzyme i always check it by electrophoresis and when i get it cut properly then i go to dephosphorylation step. so think the problem is in my dephosphorylation tecnique.

i cut my vector with stuI, it is blunt cut
so my sample composition was
DNA 7ul (1 pmol)
CIAP 1ul (1U)
Buffer 10ul
Milliq 82 ul

incubate in 37c for 15 min and immediately in 55c for 45 min
then i add 0.5% SDS and 5mM EDTA and mix and then add Proteinase K (final vol. 100ug/ml)
then incubation in 55c for 30 min or 37c for 1 hour

then phOH:CHCl3 2 times
and ethanol ppt
and check and go for ligation

can u plz tell me where is my problem

oh i have another question

what happen if i blunt a DNA which is already blunt??
i seperate a gene by speI-XbaI cut and blunt it to insert it in my previous vector
but i am a stupid i cant remember whether i make the insert fragmet blunt or not, then i blunt it again

now i am confused where is my problem

Easier than all of these is to prepare your vector by PCR rather than by cutting an intact vector. You are guaranteed that there is no intact vector background. You can choose the cloning site and restriction enzyme targets easily.