today I come to you with an interesting challenge! Prepare your brains, take your notes and GO!
I have been working with the HIGH COPY NUMBER suicide vector pSSK10 and trying to clone two fragments within it, simultaneously. I made a very good plasmid preparation, linearized very well (double digestion using NdeI and XhoI), dephosphorilated using CIP and cut from the gel. So far, everything was perfect.
For cloning I used, as a reference, 100 ng of the linear vector. Both fragments I want to clone contain 1500 bp, which correspond to the flanks of the gene I want to further mutate.
I runned my ligations at 25C for a few hours and transformed 100 uL of E. coli SM10 lambda Pir with the whole 15 microliters of my ligation. Then, I've plated 10% of the 2YT resuspended transformation and the remaining amount onto HI/Kan plates.
While for most of the vectors and E. coli lineages I've worked in the past this would result in a rasonable number of colonies for further screening, for SM10 lambda pir transformed with such ligation mix I've got a true bacterial lawn. The next days plates were competely covered and no single colonies could be distinguished.
An important detail... It happened twice. I've used different SM10 lambda-pir chemically competent cells sources, from two different labs (So I know it is not cells fault), and I used different vector batches for cloning (so I know it is not bad preparation or contamination with circular vector).
Here it goes my challenging question that will make you think for the next few hours... what is going on? What I should do to obtain isolated colonies onto my next day plates and still ensure the obtaining of recombinant clones?
Thanks a lot guys. I'll try your suggestions and the first to help me obtaining success I'll give an Amazon.com gift card of U$ 10,00. My way to say thank you.
Edited by cerqueiragm, 26 March 2011 - 06:42 PM.