Boiling Method for Minipreps

[Klaus.Matthaei at anu.edu.au]

>In article <9307310555.AA22346 at calshp.cals.wisc.edu>,
>jchen at CALSHP.CALS.WISC.EDU writes:
>>> 
>>> 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 have been using boiling method to miniprep DNA for sequencing and it has
>> been successful.  Prepare plasmid (I used pUC) with standard method. 
>> Phenol extraction is not necessary.  Denature DNA with NaOH following
>> the procedure  of using Sequenase (2.0).  In the case of not getting enough
>> DNA, simply use 3.0 ml (spin twice), rather than 1.5 ml of culture.  pBR322
>> will probably work the same way even though I didn't try it.
>> 
>> Good luck
>> 
>> J. Chen
>
>The boiling prep is wonderful, but don't even try it using HB101 as a host. 
>Re-transform your plasmids into DH5 alpha, DH5alpha F', DH1, even XL-1 Blue,
>but you will have absolutely no success with HB101 as a host:  DNA from this
>and many other commonly used hosts (like TG1) isolated by by boiling preps will
>degrade, due to a heat resistant endonuclease.  This is not just folklore; DH5
>alpha has an endonuclease-1 mutation that seems to make it perfect for boil
>preps. OK, you will get DNA from HB101 and it will probably even cut, but it
>will give you shitty sequence.  I've used DH5 alpha and Bluescript based
>vectors for plasmid sequencing since 1988, and in the hundreds of gels I've run
>many of the really awful failures I've had have been when I scrounged someone
>elses competent cells, that were not DH5 or similar.
>
>As for the boiling prep itself, we just spin down 1.5ml O/N culture, resuspend
>in 200ul STET, (forget about lysozyme), boil 10 minutes and tip the S/N into
>200ul isopropanol.  Spin down immediately, rinse in 70% Etoh, drain, then
>resuspend in 100ul TE or water containing RNAseA.  Then sequence by NaOH
>denaturation etc.  Works everytime, PROVIDING YOU ARE USING THE APPRPRIATE
>HOST!@!!!  Using colour coded tubes (for different clone groups) and
>multipipettors, 48 minipreps can be made in less than an hour with minimal
>benchtime.
>
>-- 
>Cheers,
>
>Martin 
>
>NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
>NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
>NN  NN NN           Christchurch School of Medicine            ZZZ
>NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
>                Phone (64-3)364-0880  Fax (64-3)364-750


I agree with the above.  Choose your host carefully.  Retransform if you must.

MC1061.1 is also not a good host for boiling preps.

Cheers, Klaus

(Greetings Martin)
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"I know that you think that you think that you understand what I am saying."
"But what I am saying is sometimes not actually what I mean."
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