Question about genomic library construction

Wilson netson at uxmail.ust.hk
Wed Feb 26 21:38:00 EST 1997



On 26 Feb 1997, Alexander N. Kukushkin wrote:
> >   Wilson  8~O
> >
> CIP may be serious reason for the low ligation efficiency.
> Alex

Hello Alex and everybody,

Could you suggest me how to improve the situation?  Here is the
information about my dephosphorylation:
Linear DNA (5ug) was dephosphorylated by 2U of CIP in the 100ul reaction
volume.  After dephosphorylation, 10ul 0.5M EDTA was added and incubate
the mixture in 65C for 15 min and then phenol/chloroform extraction with 3
times.  Finally, the dephosphorylated linear DNA was separated by agarose
gel electrophoresis and recover the DNA by GeneClean.

The problem of low ligation efficiency caused by CIP may be the CIP
tightly bind to the terminal of the linear DNA and inhibit the ligation.
Is the statment is true or any other reason to explain the problem?


Wilson 8-O 




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