>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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