DNA transfer

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Sun Jan 15 22:52:43 EST 1995


>pmiguel at bilbo.bio.purdue.edu wrote:
>>   I've noticed that alkaline transfer moves the DNA much faster than SSC.
>> In fact, using .4 M NaOH downward (from a foam sponge soaked in .4M NaOH)
>> DNA is totally gone from the gel (maybe 5mm thick) in less than 1 hour --
>> including 23 kb dna from my lambda/HindIII MW standard, WITHOUT
>> DEPURINATION.
>[...]
>>   BUT NOTE WELL THERE IS A MAJOR DOWN SIDE.  The DNA doesn't stop at the
>> nylon (MSI .4 um pore, non-charged).  It keeps going.
>
>I guess 0.4 M NaOH is way too much. Chomczynski (Anal. Biochem. 201,
>134-139) has used 8 mM NaOH, 3 M NaCl, 2 mM Sarcosyl. In my experience
>(with RNA, not DNA) this transfer solution gives good results.
>
>--Cornelius.
>
>--
>/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany  */
>/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795  */
>/* "Science is the game you play with God to find out what His rules are." */


I douby that 0.4M NaOH will cause this problem.  The original Reed and Mann
NAR 1986 describing alkaline blotting used this concentration although
lower concentrations will also do.  I hope that you are using a charged
nylon menmbrane since uncharged nylon does not retain the DNA as you
describe.

Cheers, 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   |
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                        "Sometimes I'm the Louisville slugger"
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