Boiling Method for Minipreps
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.
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
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.
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! ***
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