Ligations..im not getting a happy "sticky" ending :( - (Mar/06/2012 )

Hi all!

I have been recently trying to establish a stable cell line. My gene of interest is expressed in a vector that carries a neomycin selective marker. thats no good to me as my cells are resistant to G418 so im subcloning it into a different vector, pIRES puro.
My cloning strategy is cut desired vector and insert with EcoR1-BamH1, SAP treat cut vector and ligate with t4 ligase (fermentas). Sounds fine and do-able, however I have spend the last month working on this and coming up blank each time. I feel it is the ligation stage that is letting me down every time. I ligate for 30mins at room temp (as per companies protocol) and I never use more that 60ng of DNA in the overall reaction (i was told too much DNA would have a negative effect on the reaction)

it was also suggested to try and incorporate a puro site into my own pcDNA3 vector. Does anyway have an effective strategy for this?

Appreciate some advice!

Is your ligase buffer aliquotted and a fresh tube used each time? The ligase buffer contains ATP which is used to provide the energy for the ligation, and ATP degrades quite quickly.

Hi Bob1,

Yes the ligase buffer is alliquoted and it is also a freshly purchased vial and so is the t4 ligase.

Have you tried to ligate over night at 16°C? What about the transformation? What cells do you use for transformation and are they still compentent?