Question about genomic library construction
xiaohan at copland.udel.edu
Mon Mar 3 17:44:47 EST 1997
In article <Pine.GSO.3.95L.970226231323.22311B-100000 at uststf1>,
Wilson <netson at uxmail.ust.hk> 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?
Here are some tips you may want to try:
1) High competent cells. Follow D. Hanahan's protocal. You could get as
high as 10 to the 8th transformants per microgram of plasmid DNA.
2) Try double digestion instead of single digestion of your DNA. Double
digested vector will sure prevent self ligation in a quite efficient way.
3) Try your library ligation overnight at 16 degree bath. At least that is
the way I usually do.
4) When you transfrom your ligation, make sure only take 5 ul of a
20 ul ligation. If you tranform the whole 20 ul, it will drop the
transformation efficiency by 10 to 100 fold. Or, if you'd like to
transform the whole thing, clean them up before you do so.
Hope this will help. If you have any question, I will be glad to answer.
Department of Biology
University of Delaware
Newark, DE 19711
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