Question about genomic library construction

Huang Ke-xue khuang at CHEMVX.CHEM.TAMU.EDU
Wed Feb 26 22:50:52 EST 1997


At 06:41 PM 2/26/97 -0000, you wrote:
>>           Hello everybody, I have some trouble in my project and ask for
>>   your suggestion.  I have constructed a genomic library for half year,
>>   however, I still cannot clone any fraction of insert into the vector with
>>   high efficiency.  Here is the method I used:  the vector pUC19 digested by
>>   EcoRI and dephosphorylated by CIP (one hour), the insert was obtained by
>>   EcoRI digestion of the genomic DNA and the digested DNA was separated by
>>   eight fraction (the size from 0.5kb to 6.0kb).  The vector and the insert
>>   was ligated by T4 ligase at R/T for 2 to 4 hours.  After transformation, I
>>   can find normally ten to twenty colonies from the plate and after
>>   analysis, only one recombinant can be found from 16 colonies.  The
>>   ligation efficency is very low, What should I do?
>>
>>   Wilson  

It is not so easy to construct a good library. I think there are some basic
works you should try.
(1). Use the CIAP treated pUC19 and a 6.0 kb (or bigger)EcoRi fragment(any
DNA you can get like lamdaDNA) to see how many white clonies you can get.
(2). Make sure your compentent cell are realy good (check the tranfromation
efficency by pUC19).
(3).Try electroporation.
(4). Make sure your genomic DNA is really clean and high quality.

Hope this help!

Huang




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