Question about genomic library construction

Wilson netson at uxmail.ust.hk
Wed Feb 26 10:25:30 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






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