Question about genomic library construction

Alexander N. Kukushkin kan at mmcd.cyt.ras.spb.ru
Wed Feb 26 13:41:22 EST 1997


>           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  8~O
>
CIP may be serious reason for the low ligation efficiency.
Alex



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