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Boiling Method for Minipreps (CTAB variation)

Dennis J. Templeton djt2 at po.CWRU.Edu
Tue Aug 3 10:20:16 EST 1993


In a previous article, neale at mbcf.stjude.org () says:

>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.
>> (deleted>>
>
>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
>
>
Our normal MP uses HB101 with a boiling/CTAB protocol taken from a 
Biotechniques article.  We too noticed the heat stable nuclease that Geoff
mentioned above, but didn't want to forego HB101's.  The nuclease seems to
be eliminated by the CTAB procedure.  Overall, the method takes much less
time and $$ than magic miniprep$.  20 MP's takes me about 60 minutes, off
and on time.

Have fun.
dennis

Boiling/CTAB minipreps

Grow HB101 in LB amp (8ml) overnight.  Spin 3-5 k 10 mins, drain. 
Resuspend pellet in (for each tube) 0.3 ml STET (8% sucrose 50 mM Tris 8.0
50 mM EDTA, 0.1% Triton (not 5% as Maniatis says)) + 10 ug RNase A and a tiny 
pinch of lysozyme... (about 1 ug per tube).  

Transfer to a microfuge tube. No deliberate incubation is needed.  Boil 1.5
minutes, spin 15,000 rpm 10 minutes, pluck out pellet with toothpick.  Fill
tube with 0.5% CTAB in water (warm to make sure it is dissolved) from a squirt
bottle, about 1 ml. (final CTAB then about 0.4%).  Mix well, spin 5 minutes
15,000.  Pour off sup and let drain a minute.  

Add 250 ul 1.25 M NaCl, put in vortex mixer for 3-5 minutes (pellet is
brownish, and is frequently at the sides of the tube, near the top.  The
high salt re-dissolves the CTAB/Na precipitate)  

Fill tube with 95% EtOH (squirt bottle) and mix well for 30 secs or so. 
Spin 2 minutes 15,000, pour off sup, and rinse tube with 95% EtOH (squirt
bottle again) to remove high salt.  Drain well for 5-10 minutes.   

Redissolve in 50 ul TE, put on vortex mixer for 2-5 minutes.  Yield (for a 
5 kb pUC-based plasmid) is about 0.5 ug/ul from HB101. There are RNase 
digestion products, but much less than in most MP's  When we get a positive, 
we then phenol/chloroform extract this DNA and reprecipitate  before 
proceeding on with the next part of the construction. We also do this (and 
precipitate from high ammonium acetate) for sequencing.  We sometimes scale 
this up to 300 ml for a "maxi" plasmid prep, but overall we have stayed with 
CsCl for large scale.        

This MP DNA is fine for mammalian cell transfection and lipofection too.



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