Sub cloning

Marieke R. Koedood Zhao rkoedood at bu.edu
Wed Aug 6 10:54:19 EST 1997


We had similar problems with and 'unclonable' cDNA. But we conquered it.
Took 2 months and a lot of effort though. Here is what we did that 
eventually got us the clone. First an interesting observation that you 
might want to check out - since it was my student's first cloning, I let
her do the insert alone as ligation control too. To our surprise, the 
insert alone gave loads of colonies, as many or more as our vector+insert
plates. Several repetitions convinced me that she didn't switch tubes
and there is indeed something in our insert-prep that transforms quite
efficiently. some suggestions that we collected was that it is 'super-super-
coiled' (form V DNA, but don't ask me anything more about this) that runs 
faster than supercoiled. THis is supposedly denatured DNA, runs faster
than sc, co-migrates with our beautiful fragment, is resistant to cutting
with enzymes and cannot be purified away.

Doing numerous ligations, we always got the high insert background, little
or no vector background and upon doing minipreps we don't get anything 
bigger than the vector alone, but plenty running like the plasmid the 
insert came from.

How did we finally conquer it - by doing colony lifts of the plates and
hybridizing with a part of the insert fragment (made from another, unrelated
prep). This nailed down something like 15 out of about 100 colonies as 
having the insert. Of these we got 3 with inserts, but in the wrong 
orientation. We do suspect that our expression plasmid with the correct
cDNA is somewhat toxic and that the bugs either don't grow, or recombine 
some of the insert out.

To increase our odds, we did 5 independent ligations, resulting in >500
colonies, did colonie lifts, giving us <20 colonies for minipreps. Out
of these there were 5 bigger than the vector, and only one with the insert 
in the correct orientation. So, we managed to beat the odds. Hoping that
the bugs didn't do something to make our beautiful clone ineffective...

Hope our story of cloning woes is somewhat informative....

Marieke


L.D. Ofner (BMBLDO at leeds.ac.uk) wrote:
: Hi everybody,

: I've been trying to sub clone a full length cDNA sequence (4.5kb) into an 
: expression vector for ages now with no luck and it's driving me mad ! There 
: are EcoR1 sites at either end of the insert in its original vector which I'm 
: using to cut it out (there are no other restriction enzymes that I can use 
: for directional sub cloning which won't cut the insert sequence or the 
: expression vector in the wrong place). I've performed the necessary 
: restriction digest for cutting the insert out and cutting the expression 
: vector, purified both from agarose gel using Wizard clean up and 
: dephosphorylated the expression vector ready for ligation. I've set up 
: numerous ligations with differing ratios of insert:vector (including having 
: insert in vast excess) and have had loads of colonies growing on plates. But 
: when I prep up the DNA ( usually pick at least 10 colonies) and check by 
: restriction digest, none seem to contain insert ! I've checked that the 
: vector is dephosphorylated by tranforming my competent cells with it and I 
: get no resulting colonies on plates. Help ! Where am I going wrong? I'd be 
: grateful for any advice or suggestions (soon before I go totally insane !).

: Thanks. Lisa. 



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