Plasmid Prep for Sequencing (Sorry if this is FAQ)

Brian Foley brianf at med.uvm.edu
Thu Jan 13 11:51:19 EST 1994


Hinayana Bawagan (bawagan at umdnj.edu) wrote:

:  Dear netters,

:      What is a reliable DNA prepn method for sequencing?  I am sure
: that most of you have discovered little fine-tuning steps that are
: critical to the conventional protocols of alkali lysis or boiling.
: Thank you very much.

       There have been so many answers to this question posted to
Bionet.molbio.methods-reagents, that it is really worthwhile to
use GOPHER or another net tool to look through the archives of
this list at IUBIO.  Use gopher to navigate to Indiana, IUBIO, 
net-work news, search network news archives at IUBIO.  Then
search for "sequencing minipreps" or just "sequencing".

 --
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
********************************************************************

      Below is a small sample of just a few of the tips you will find.

***********From the IUBIO archives of METHODS-REAGENTS***************

> 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.
........
> -- 
> 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
>

Well, I have been using HB101 for a while.  It works just fine.  Here is
the trick: Denature your plasmids as soon as possible and keep the dry
tubes
at -20C for future use.  If the endonuclease really bothers you, a phenol
extraction will take care of it.  As a matter of fact, I prefer HB101 as a
host because cells are easy to resuspend into the lysozyme buffer.  This
makes
a big difference when you do a large number of minipreps.  Good luck!

JC

jchen at calshp.cals.wisc.edu
Department of Food Microbiology and Toxicology
University of Wisconsin
Madison, WI 53706
*********************************  2  *****************************
>>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

************************************  3  ***********************
>As a matter of fact, I prefer HB101 as a
>host because cells are easy to resuspend into the lysozyme buffer.

Somewhere in my collection of references there is a report about lysozyme
not being necessary, and even reducing plasmid yield, in some strains of
_E. coli_.  So I could not help wondering if anyone has tried a boiling
miniprep without adding lysozyme.

I personally use the alkaline lysis method modified somewhat, but without
lysozyme, for mini-, midi- and maxipreps of plasmids.

Good luck,

Jim Owens


************************************  4  ***********************

> jchen at CALSHP.CALS.WISC.EDU writes:
>>As a matter of fact, I prefer HB101 as a
>>host because cells are easy to resuspend into the lysozyme buffer.
> 
> Somewhere in my collection of references there is a report about lysozyme
> not being necessary, and even reducing plasmid yield, in some strains of
> _E. coli_.  So I could not help wondering if anyone has tried a boiling
> miniprep without adding lysozyme.

> Good luck,
> 
> Jim Owens

Jim, 

We dropped lysozyme out of our boiling mini-prep procedure a year or two
back. 
We always use Bluescript/DH5 alpha combinations, and it works fine.  There
was
a suggestion that the yields were slightly down in the absence of
lysozyme, but
we still get plenty of DNA (ca 10ug per 1.5ml O/N) and it sequences fine. 
This
gives us a really minimal protocol, especially as we use a multiple
dispensor
(Eppendorf with Combitip attachment) for the STET and ethanol steps.  I
feel
the Bluescript yields are better than pUC, and certainly much better than
pBR322, so I can't vouch for not using lysozyme in all plasmid/host
combinations.  Incidentally, I seem to recall the original Holmes and
Quigley
paper stating that the lysozyme was only present to help bring down the
lysed
cell debris etc during the centrifugation.


-- 
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

************************************  5  ***********************

In article <1993Aug4.221947.9602 at alw.nih.gov>, Jim Owens
<jow at helix.nih.gov> writes:
>In article <9308032222.AA20703 at calshp.cals.wisc.edu> ,
>jchen at CALSHP.CALS.WISC.EDU writes:
>>As a matter of fact, I prefer HB101 as a
>>host because cells are easy to resuspend into the lysozyme buffer.
>
>Somewhere in my collection of references there is a report about lysozyme
>not being necessary, and even reducing plasmid yield, in some strains of
>_E. coli_.  So I could not help wondering if anyone has tried a boiling
>miniprep without adding lysozyme.
>
>I personally use the alkaline lysis method modified somewhat, but without
>lysozyme, for mini-, midi- and maxipreps of plasmids.
>
>Good luck,
>
>Jim Owens


As a matter of fact I just did that experiment today because we were
also discussing whether or not it was important.
Our result was significnalty higher DNA yield when lysozyme was added.
this was using strain TG1 and high copy plasmid pBluescript.
I strongly suspect the answer is strain dependent, but for TG1, which is
a very hardy strain, lysozyme really helped.


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