Q about boiling miniprep sequencing.

David Micklem drm21 at mole.bio.cam.ac.uk
Mon Mar 20 07:41:26 EST 1995


In article <3kcl99$14qm at news.cuny.edu>, kang at msvax.mssm.edu wrote:

> Hi colleagues;
> 
> I really really like boiling miniprep methods.

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

Here I must disagree.  Its good for all four - see below.
> 
> 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.

Cool! How?  I can manage about forty an hour, but its really boring (and
when do you ever need to do more than forty).  WOuldn't this kind of thing
be perfect for a robot system?

 >However, the problem I 
> encounter is I can not use this method for dye terminator cycle 
> sequencing unless doing the phenol extraction.

Why do cycle sequencing? Taq is a lousy enzyme.  Really fussy about
template, buffer conditions etc.  Look how hard PCR can be if you don't
optimise it.  AND it shows significant and annoying sequence-dependant
peak-size variability. 

In my experience Sequenase has none of these problems.  Sure, you need
more template, but what else are you going to use that miniprep for?  You
can easily get two sequencing reactions, several digests and a cloning
step from a single miniprep, and its easy to make more! The Sequenase
Terminator ds and ss sequencing kits from ABI work really well, need a
much simpler clean-up procedure at the end, and are no more expensive/rn
than the Taq ones (at least at the prices I've seen). Especially as you
can squeeze at least half-as-many-again reactions from the kit as they say
- at the expense of a more hassle-full protocol.      

If anyone knows if you can just cut down on the amount of terminators you
use, do let me know.  On a similar note, is it possible to buy just the
terminators (cheaply) and make up the other components yourself?

> I heard that Millipore 
> PVDF membrane filter unit can absorb proteins while passing 
> through double strand DNA. Is there anybody who tried that 
> method?

Not me, but if you're going to go the expensive-membrane route, why not do
the whole DNA-prep that way and buy kits?

> Or do you know some methods can be used for cleaning boiling 
> preped DNA other than phenol extraction? (phenol ext. is my last 
> choice)

Yes, PEG pptn.


FWIW I've been getting around 300bp by manual sequencing, and 350-450 by
automated sequencing.

Cheers, David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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