Gateway Cloning Problem - Lawn of E. Coli Cells - (May/01/2009 )

I'd really appreciate any advice I can get on the problem I and another lab mate are having.

Initially, I amplified the desired cDNA using the Platinum Pfx PCR kit (Invitrogen) and confirmed size on a gel, no other bands were observed except desired so I didn't do any additional purification steps. Currently, I have cloned my PCR product using the PentrD/TOPO kit (Invitrogen) into Top10 E. Coli to generate my entry clone. After confirming through sequencing that the clone was correct I moved on to perform the LR Clonase reaction (Invitrogen). I have been performing 1/2 or 1/4 reactions. I let the LR reaction incubate for 2 hours, transform in to Top 10 cells and plate on LB + AMP. Everytime I complete the transformation I end up with a lawn of E. Coli. This is very frustrating as I have double checked the ccdB sensitivity, as well as a few other things and can't come up with any idea as to what is going wrong. I am currently going to try plating a reduced amount of the transformation. If anyone has any other thoughts I would appreciate!

Thanks.

Have you checked your vector that you are moving it into? If the vector has already been used for a previous LR reaction and and you might be using some derivative of that, this is possible.

scolix on May 1 2009, 11:32 PM said:

The vector was from a glycerol stock, specifically I am using PGADT7GW and PGBKT7GW. I just did a fresh mini-prep on that the other day. It may just be the transformation efficiency of the Top 10 cells. Either way I am getting a very low yield of colonies with the actual insert.

hi,
did you check the antibiotics? that might be a simple reason..

Exactly same problem!!

Same vectors (entry and destination) and same problem. If you find out the solution please make a post. I am very frustasted with this cloning!!

I think the destination vector is not stable.

Please, feedback the solution to the problem.

Thank you in advance!!