Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Aug 16 05:39:12 EST 2000
In article <399A2892.C9741DB7 at students.bendigo.latrobe.edu.au>, Julie
Thomas <ja6thomas at students.bendigo.latrobe.edu.au> writes
>suspect my DNA is not resuspending well. I usually heat it at 37oC for
>1/2 hour, as well as leaving it at least overnight. Has anyone else
>have this problem or any ideas how to combat it (I'm not sure if I
>should heat the DNA to a higher temp., leave it longer, all I know is
>that I can't vortex the tubes as it is likely to cause shearing).
What length is the phage DNA and what sort of concentration are you
trying to suspend it at.
I have mg's of T5 (at 100kb or so) that is completely in suspension at
2.5mg/ml but it is very very viscous and took days to dissolve in TE at
4C. I've never tried using higher temp. Although the phages we do for
internal use go through several phenols I am still paranoid about
resuspending a few hundred mgs of several days worth of work at 37C!
Probably 'cos years ago I lots a large batch during dialysis against TE
(after the phenols) at room temp. overnight!
As for shearing. I know I can vortex lambda and T5 (only ever tried on a
small scale) and I've never noticed any shearing. I would try and use a
gentle very slow swirling shaker or one of those rotating/inverting tube
mixers on very low speed.
Alternatively your DNA still has a lot of protein with it or you
overdried it at the ethanol stage. I never worry unduly about having
some 70% ethanol left on the spooled DNA. As there is usually 100mg plus
of DNA going into say 100ml of TE I've never found that a few 10's of ul
of 70% ethanol has any effect on the usage the DNA is put to (screening
for RE's or monitoring RE activity during purification).
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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