AAARGHHH, cloning driving me insane, HELP PLEASE! - UPDATE on same 5' and 3' ends (Jan/25/2010 )
OK,
I posted a topic a couple of weeks ago, asking how there can be a bias on insertion of a fragment that has identical 5' and 3' ends (cut by EcoRI). I was consistently getting the fragment in reverse orientation, if inserted at all. I then did >200 colony PCRs and ended up with 7 colonies that seemed to have the insert in the correct orientation. I tested by restriction digest (just a BamHI digest which gave a ladder of bands at the correct expected MWs).
It turns out I cheered too early, as now that I have sequenced those promising constructs, using a vector specific primer, none of them turn out to have the insert at all... 
I do not understand what is going on....
For those who need more numbers:
Vector is 10.9kB (9kB+ other insert of 1.9kB), insert is 3.8kB.
Out of the colony PCR experiments I got 8/40 with insert, 7/>200 with insert in correct orientation.
My boss is now telling me to start at square 1, and PCR amplify the whole gene of interest (5.7kB) with new primers (and new restriction sites making the cloning steps directional this time), subclone into a TOPO vector and clone by restriction cloning into the destination vector. I am a little sceptical.... If the problem is that the bugs do not like the gene's DNA, would I not run into the same problem if I start anew? In addition I wonder how easy it will be to PCR up the whole 5.7kB of this gene...
Would anyone have any advice on how to go about getting this cloning to work???
Hello flygirl on Jan 25 2010, 02:14 PM said:
Sorry to hear about your cloning dramas. I missed your original posts (on holiday) but IMO amplifying the gene with new RE sites is easy and won't take too long. Get someone to double check your primers etc before you start.
Clare
I posted a topic a couple of weeks ago, asking how there can be a bias on insertion of a fragment that has identical 5' and 3' ends (cut by EcoRI). I was consistently getting the fragment in reverse orientation, if inserted at all. I then did >200 colony PCRs and ended up with 7 colonies that seemed to have the insert in the correct orientation. I tested by restriction digest (just a BamHI digest which gave a ladder of bands at the correct expected MWs).
It turns out I cheered too early, as now that I have sequenced those promising constructs, using a vector specific primer, none of them turn out to have the insert at all...
I do not understand what is going on....
For those who need more numbers:
Vector is 10.9kB (9kB+ other insert of 1.9kB), insert is 3.8kB.
Out of the colony PCR experiments I got 8/40 with insert, 7/>200 with insert in correct orientation.
My boss is now telling me to start at square 1, and PCR amplify the whole gene of interest (5.7kB) with new primers (and new restriction sites making the cloning steps directional this time), subclone into a TOPO vector and clone by restriction cloning into the destination vector. I am a little sceptical.... If the problem is that the bugs do not like the gene's DNA, would I not run into the same problem if I start anew? In addition I wonder how easy it will be to PCR up the whole 5.7kB of this gene...
Would anyone have any advice on how to go about getting this cloning to work???
I can feel your pain. Single site clonings can indeed go silly sometimes. Why not use "colony-cracking" instead of a pcr screen? What you could do is pick-up 50 colonies or so, dunk each in 20ul of LB (take 1ul of this for growing a culture o/n for miniprep), treat the remaining with cracking buffer and just run it in a gel. Samples with inserts will show slightly slower migration as compared to the rest. You could then miniprep only those samples for further tests.
I think your boss has a point, may be you can consider his approach as a backup while you go back and troubleshoot your method (may be you are not CIPing your vector enough?). Anyway, 5.8 Kb should not be a problem at all if you use Phusion Taq (a few days ago I pcr'd a ~9.5 fragment with it, my highest so far). If you use Phusion, design primers to include appropriate restriction sites and then digest the pcr product before inserting it into your vector. Hope that helps.
