Dear Keyboard Watchers:
I posted this a few days ago, but I forgot to specify what subject. Here
is the original posting:
I saw people asking questions about rapid preparation of DNA for
sequencing. Here are a few tips that should help to solve your problem and
to save your money.
1. Do your mini-scale plasmid DNA isolation from E. coli as described by
Maniatis et al. (1982).
2. DNA from 1.5 ml of cells is dissolved in 40 ul of TE buffer and
purified further by precipitation of the residual RNA and proteins with
addition of 20 ul of 7.5 M ammonium acetate on ice for 5 min (note!!
precipitation of the residual RNA and proteins in a large volume is not
effective; although some people normally use ammonium acetate for their
minipreps, that is not good enough). Following centrifugation for 2 min,
DNA in the supernatant is precipitated with 2 volumes of ethanol. DNA
prepared in this way yields superior DNA sequencing results.
1. Do not over-grow your cells (the length of time is strain-specific)
and do not use old cells (even store at 4C)
2. The slow-growing stains (such as XLblue or DH5-alpha) normally produce
better DNA for sequencing.
3. Acrylamide solution is stable for a long time as long as you do not
mix it with urea and TBE.
Please try it and inform me about the above tips.
For experts, I have to say that I am sorry that I waste your time.
Good luck to everybody.
The Research Institute
Hospital for Sick Children
e-mail: jhu at sickkids.on.ca