Boiling Method for Minipreps

John Nash nash at biologysx.lan.nrc.ca
Fri Jul 30 19:08:04 EST 1993


In article <23c14a$r60 at samba.oit.unc.edu> tencza at med.unc.edu (Michael G. Tencza) writes:
>From: tencza at med.unc.edu (Michael G. Tencza)
>Subject: Boiling Method for Minipreps
>Date: 30 Jul 1993 20:42:50 GMT

>I would like to know if anyone uses the boiling method for plasmid
>minipreps to prepare DNA for sequencing.  I wish to try this method for
>several pBR322 constructs I have recently transformed into HB101.  Any
>hints would be appreciated.
>^X

I use the boiling method for all my screening-sequencing... no phenol, no 
RNase, no glass milk.  Use Terrific Broth to grow the cells, spin down 1 ml 
cells, resuspend in 300 ul STET + 30 ul lysozyme.  Boil 40 sec, spin 30 min 
and pull out pellet with toothpick.  Isopropanol ppt, wash with 70 % EtOH, 
air dry and resuspend in 40 ul TE.

To sequence, take 8 ul plasmid prep, and do a standard NaOH denature 
EXCEPT do it in the presence of the primer and incubate in a boiling bath 
for 2 min.  The argument is that hot alkali degrades the RNA, so no need for 
RNase.  Then, just sequence as usual... I could dig out the reference if you 
want.

One thing... I use a pUC derivative, I wonder if the pBR322 constructs would 
have a good enough copy number for this technique... I know the replicon's 
the same, but I think pUC still has a higher copy number.

  cheers, John

  John Nash                           | Email: Nash at biologysx.lan.nrc.ca.
  Institute for Biological Sciences   |
  National Research Council of Canada | Email to my other NRC accounts
  Ottawa, Ontario, Canada.            | is usually forwarded here.
	  *** Disclaimer:  All opinions are mine, not NRC's! ***



More information about the Methods mailing list