digestion of genomic DNA from mouse tails

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Thu Nov 3 16:30:02 EST 1994


>I have problems in digesting genomic DNA from mouse tails. I use the
>standard protocol: 1x phenol, 1x phenol/chloroform, 2x chloroform, EtOH
>precipitation, spooling of the precipitated DNA and resuspension in TE.
>Does anybody have experience in the matter and could help me solve this
>problem?

We use the following for PCR but the DNA cuts well for Southerns as well.

PREPARATION OF TAIL DNA

1.      Mark ears and record in register.
2.      Cut tails (~0.5cm) into 500 =B5l lysis buffer (add 250=B5l=
 proteinase k
to 25ml buffer)
3.      incubate at 56=B0C overnight with agitation.


LYSIS BUFFER:

=46inal conc.     Stock Solution  ml/litre

TrisHCl  100 mM 1.0 M   100 ml
EDTA  5 mM      0.5 M   10 ml
SDS  0.2%       10%     20 ml
NaCl  200mM     2.0 M   100ml
Proteinase K 100=B5g/ml   10 mg/ml        100ml

Make up to 900 ml (omit Proteinase K) with milliQ water and filter sterilise=
.
Aliquot into 25 ml and store at 4=B0C
Add proteinase K just prior to use (stored frozen).

DNA Preparation.
1.      Vortex tube.
2.      Centrifuge tubes at 6,000 rpm (or harder) for 10 minutes. (PCR for
single copy genes can be performed directly on 1 =B5l of this prep if stored
at 4=B0C).
3.      Pour supernate into tubes containing 500=B5l isopropanol, mix well
and fish out the DNA using a sterile pasteur pippette.
4.      Rinse with 70% ETOH
5.      Dry DNA in vacuo for 10 minutes (do not speedyvac).
6.      Redissolve DNA in 600=B5l T10 E0.1 by heating to 65=B0C for 30 minut=
es.
7.      Measure the OD at 280 and 260 nm.
8.      We dilute 5=B5l of this preparation with 45=B5l T10 E0.1 and use 5=
=B5l of
this (~100ng DNA) for PCR of single copy genes.
9.      Store solutions at 4=B0C (Do not freeze).

If you are paranoid you can give this DNA an EtOH pptation before restrictio=
n.

Good Luck, Klaus

*************************************************************************
Klaus Matthaei
Head, Gene Targeting                            |           |
The John Curtin School of Medical Research      |  _--_|\   |
The Australian National University              | /      \  |
Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
Landline: 61 6 249 3782
                        "Sometimes I'm the Louisville slugger"
                        "Sometimes I'm the Ball"
                                Dire Straits
*************************************************************************






More information about the Methods mailing list