mn5y@krebs.acc.Virginia.EDU (Mukund Nori) (09/27/90)
This is just to say thanks to all those who e-mailed me suggestions for
gfetting my ligations to work again. The improtant lessons I learnt are:
1) do not digest the DNA for more than 1 hour; longer times decrease
ligation frequencies considerably.
2) it is prefarable not to stain the gel with EtBr; use UV shadowing to
visualize the band, mark it an then cut it out under regular light not UV,
thus reducing the exposure to UV.
3 preferable not to freeze the gel piece during recovery of the DNA from
gel.
4) clean several times with EtOH, just to be sure that there is no phenol or
chloroform, etc. left.
5) use fresh ligation buffer.
6) preferred conditions are 15C for 16-24 hours (at least for mine).
7) easy check by running a minigel and looking for high MW bands.
If this helps any others, I will be gratified. My thanks again to all. I
find it easier to post this than reply individually.
Take care.
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___Raistlin___ Mukund Nori
Raistlin@Virginia.EDU mn5y@krebs.acc.Virginia.EDU
"VIOLENCE IS THE LAST RESORT OF THE INCOMPETENT" Asimov