Sub cloning

hadi al-hasani hadi at hatespam.helix.nih.gov
Mon Aug 4 23:32:14 EST 1997


Hi Lisa,
I have had the same problem with an insert which was toxic for the
bugs when cloned in one direction into the Bluescript KS - but not in
Bluescript SK...
I suppose you need to know if indeed your insert is toxic. (If it's
toxic you'll ligate until the end of time...)

Try the following: 
Make primers upstream and downstream of your MCS, respectively (or use
your sequencing primers if you have any) and run a PCR with your
ligation reaction. If you get your 4.5 kb product you definitely loose
your ligation products. If you don't see any "insert" band something
is wrong with your ligation. Don't forget to include a control to see
if you can amplify xxx kb with our primers.
However, my guess is that the toxicity is pretty unlikely to happen in
both orientations of your insert. You might try to  fill in the ends
of your EcoRI or whatsoever ends and ligate it blunt end. This might
not change very much but adds some kind of variation and at least you
might have some more fun in going in sane...

P.S.: I'm still stuck with this (...) subcloning step for 2439 other
reasons...

Hadi
NIDDK/NIH
remove "hatespam." if you prefer to have a valid email address



On Fri, 1 Aug 1997 14:28:01 +0100 (BST), BMBLDO at leeds.ac.uk (L.D.
Ofner) 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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