HELP! - cloning problem

Bernard P. Murray, PhD bpmurray*STUFFER* at
Wed Nov 4 19:19:48 EST 1998

In article <00075053.1556 at>, MSRA at (MSRA) wrote:

>      Hello,
>      We have a strange cloning problem (and totally frustrated!).  After 
>      the blue/white selection, we tried insert check by colony PCR.  Here, 
>      most of the case, there is no band amplified, even though we selected 
>      white colonies.


>      Our vector is 5-6 kb (pBAD and pMAL)

If "pMAL" is NEB's vector for fusion expression I am pretty sure
it does not contain LacZ so you can't do blue/white screening
(*all* the colonies should be white).  I'm not familiar with
pBAD but it could be the same situation.

PCR screening is pretty reliable in my hands.  Did you have
a good positive control?  Are your primers for the plasmid
or for the insert?  Are you swamping the reaction with
contaminating empty vector or free fragment?  

How did you clean up the fragment?  Did it look clean on a gel?
Did you check your cut vector after digestion (and cleanup)?

It sounds as if you just have to go back and pick more colonies.
Try and make the ligation as efficient as possible.  Try a ligation
in the absence of insert to check your background.  If your colony
PCR works on a positive control then just keep at it until you see
something with an insert.  If pMAL is what I think it is it should
have a built-in LacIq and so should not necessarily need a LacIq
host strain (otherwise I would suggest reslecting for the F' in
your JM109 or moving to a strain with a selectable F' such as
XL-1 Blue).
     You are not really pushing the envelope for Big Plasmid +
Big Insert but the efficiency will certainly be a lot lower
than with a 3 kb cloning vector.  Just go for brute force and
fill up the PCR machine, or try colony hybridisation with your
insert, or switch on the expression of your insert cDNA and
screen with an antibody etc. etc.
     Good luck,
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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