Gateway Blunt end cloning - no colonies at all (Jul/23/2005 )

Hi, I've been reading the Molecular Biology topics, and there are a couple of persons with problems with Gateway.

I have also a problem. I am using the Vector Conversion System kit, to convert my expression vector into a Destination vector, ready to recombine later on with the Entry vector carrying my genes to express.

My problem appears when trying to ligate a blunt-end cassette from the kit (1,7kb) into my vector (5.7kb).
I had dephosphorylated the vector, and also added PEG to the media.

Any advice or self experience on this technique?
How much PEG should be present in the reaction?

Thanks

M

-marcfe-

QUOTE (marcfe @ Jul 24 2005, 08:17 AM)

which companys ligase are u using for blunt end ligation?
i tried using MBI fermentas . they give 50% PEG in separate eppendorff.
if ur volume is 10microliter , u can use the 50% PEG in the final concentration of 5%
anyways if u order the product u can get the insturctions along with it.
plus u have to use more amount of ligase and dna

hope this helps

-phytoviridae-