HELP: Ligation

Nicolas Lemee lemee at ibm.net
Fri Sep 25 19:54:34 EST 1998


Do you phenol/chloroform your PCR before digesting it ?
I ph/chl twice then ethanol precipitate before digesting.
Works like a breeze for me.
Also do you confirm that you still have DNA after the gel clean ?

--
Nicolas


>I have a problem with cloning a PCR amplified fragment into a
commercial
>expression vector. The PCR amplicon has the same Restr Enz site and the

>plasmid only one site ofr it. i end digest the plasmid and fragment,
>dephosphorylate, separate on a gel, and purify the gel bands (kit) and
then
>use the fragments for ligation (20h, 4/ 16 deg C). Once I een saw a
>presumably ligated fragment, but no transformed colonies of XL1 Blue.
> suggestions, tips, tricks etc?




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