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
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