3 replies to this topic

[wizzkid]

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!

[bob1]

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.

[wizzkid]

Hi Bob1,

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

[Papaver]

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?