I have been struggling on Gateway LR reactions. Here is the situation:
(1) Suceeded in BP reaction, so got inserts (~400bp long) in DONOR vector with L sites.
(2) Des vector has two pairs of R sites and it is 24.5 kb long. Originally thought LR kit should be easy to replace the ccdB sites (flanked by attL sites) by inserts in DONOR vector.
However, after LR raction and transformed DH10b cells, got lots of colonies. After diagnostic RE digests, 95% of colonies got undigestable random vectors. So further consulted Invtrogen, they told me that my Des vector is big and has two pair of R sites, so it is easy to have non-specific recombination events, especially in DH10b cells that have aberant recombinase activities.
So I switched to Stbl-4 cells and grew transformants in 30C to minimize the random recombiation events, now I also got lots of colonies, after RE, I did get much less non-digestable unknown vector. But from the 10 colones I digested, 7 of 10 showed only Des vector pattern and NO reacted product.
I would really need help on
(1) how to facilitate the effective LR reactions between my Des vector and inserts in DONOR vector?
(2) how to explain the majority of colonies in Stbl-4 tranformed cells to exibit only Des vector with ccdB in them? in another word, how come ccdB did not kill stbl-4 cells?
Thank you so much for your insights!