Changing the vector in the cA library

Dom Spinella dspinella at chugaibio.com
Mon Jul 7 10:56:12 EST 1997


Dooyeon Kim writes:

> Hi, everybody. it may be the double posting because of the problems in my
> news server. sorry 
> For some reasons, I have to change the vector system of my cDNA library.
> My cDNA library is inserted to pJG4-5 vector(which is a component of two h
> ybrid system) at the EcoRI and XhoI site. I want to change the vector system
> to different kind.
> I plan to do this by simply cut the library, dephosphorylate the fragments,
> and ligate to the different vector. But my colleagues disagree with this
> idea. They worried about the low efficiency of ligation. In my thought,
> This problem can be overcomed by increasing the amount of DNA, but they
> did not agree with my idea.
> It is very difficult to make a new cDNA library at my situations.
> Is there anyone who advise me about this problem?
> Any advice or information will be greatly appreciated.
> 
> Please help me

Dooyeon:

I agree with your colleagues -- your approach to changing out the vector
is sub-optimal.  For one thing, you will destroy any cDNA inserts that
happen to have a restriction site corresponding to whatever sites you
plan to use to remove the cDNA from the initial vector.  Why not simply
get a pair of PCR primers complementary to your initial vector sequences
flanking your cDNA cloning site.  You can then amplify an aliquot of you
initial cDNA library, and then blunt-clone the PCR products into your
new vector.  The new library probably won't be quantitatively
representative, but if all you need to do is to fish new genes out of
it, this should work.  Good luck. --D.G. Spinella



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