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?
>> Wilson 8~O
Get pZeroI or pZeroII from Invitrogen. You don't have to bother with CIP
treatment and I routinely obtain 90% recombinants.