Q about boiling miniprep sequencing.

kang at msvax.mssm.edu kang at msvax.mssm.edu
Fri Mar 17 13:43:53 EST 1995


Hi colleagues;

I really really like boiling miniprep methods.

It is superb in RE digestion, manual sequencing with T7 polymerase 
and subsequent cloning but not so good for automatic sequencing.

My  goal is developing a method which enables to purify 1,000 
sequencing grade DNAs per day by a person.

So far, I developed a modified boiling miniprep method by which a 
person can do 1,000 minipreps a day easily. However, the problem I 
encounter is I can not use this method for dye terminator cycle 
sequencing unless doing the phenol extraction. I heard that Millipore 
PVDF membrane filter unit can absorb proteins while passing 
through double strand DNA. Is there anybody who tried that 
method?
Or do you know some methods can be used for cleaning boiling 
preped DNA other than phenol extraction? (phenol ext. is my last 
choice)


Mine is following; By AB 373A and recommended method by them.

1. Boiling miniprep alone ------      Impossible to read signal

2. Boiling miniprep + Strata clean resin --- 150 bp but not desirable

3. Boiling miniprep + Strata clean resin + Sephadex G-50 --- little 
improvement but still not desirable. 

4. Boiling miniprep + phenol ext.--- 450 bp with some mismatches, 
almost same quality as Promega wizard miniprep. However, it takes 
some time and you can not process 1,000 samples a day.


**** If it is manual sequencing, I can read around 200 bp without 
further purification.


Thank You in advance

Chulho Kang
Wizard of TM (transgenic mice)



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