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Boiling Method for Minipreps

neale at mbcf.stjude.org neale at mbcf.stjude.org
Mon Aug 2 12:13:53 EST 1993


In article <nash.215.0 at biologysx.lan.nrc.ca>, nash at biologysx.lan.nrc.ca (John Nash) writes:
> 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! ***


I'll just add my two cents here.

 Boiling method works fine as John has written above. 

Prior to sequencing, you can also heat your DNA in alkali at lower temps
 if you extend the time of incubation eg 15-20 min at 37C.

However, the main thrust of this posting is to beware of using HB101 for
boiling prep DNA. This strain and others (check Maniatis) have a heat-resistant
nuclease that is poorly inactivated by the boiling mini-prep method.
Consequently you should avoid these strains if you use the boiling method.Your
DNA will degrade on storage (I have learned the hard way!)

 We currently use DH5alpha and DH10B strains with this method and the sequencing
quality is great, and the prep time short (faster than Promega "Magic Minis").

Geoff Neale 
St Jude Children's Research Hospital
Memphis TN



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