Hi, everyone
I am using a 4.3kb vector to clone both a promotor and an enhancer. I already have a positive miniprep with no mutations in the promotor, therefore I took some of this plasmid, digested it with BamHI, treated it with CIP and off I went trying to insert the enhancer now. I did everything as I was doing before: made sure the plasmid is digested by running some of it next to the undigested plasmid, made sure the enhancer is properly digested in both extremes (BglII) as well with a insert-insert ligation, made the math considering plasmid and insert size to get how many nanograms of each you need...
And I got zero positive colonies out of 24. Zero!
This is not what we usually get. Last time I cloned the promotor I got 3 positive colonies out of 15.
What's the big difference? Is it she size?
Vector: 4.3kb
Promotor: 1.4kb
Enhancer: 1.6kb
This means that by the time I try to insert the enhancer, the vector is only 1.3kb larger, 5700 instead of 4300. I don´t understand how that can make such a difference.
None of my inserts is huge, neither is the plasmid, I made sure they were all properly digested, I performed ligation as usual (16ºC O/N, 5ul of total volume), transformation went OK since I got plenty of colonies...
What should I try? How can I know what´s wrong?
Thank you very much in advance
It seems, you performed your cloning almost perfectly. Since you are obviously very experienced, I guess that you did not use BamHI for the analysis.
The most likely explanation is an incomplete CIP reaction. I recommend to CIP the vector again and do everything as before.
There doesn't seem to be anything wrong with your methods. Maybe check that the selection medium are fresh? Other than that, just pick more colonies to analyze. Sometimes you need to screen a few dozen, sometimes you get what you want on the first try.
Good luck!
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