cycle sequencing from mini prep DNA

Andre Hamel hamel at ccu.umanitoba.ca
Fri Apr 23 18:15:21 EST 1993


We do following regarding use of Promega's Magic minipreps:

o  Use 5 - 10 mL overnight culture ... ALL of the cells pelleted,
resuspended into 200 uL TE, etc as supplied with kit

o  Apply to cartridge as recommended by kit. Nothing different so far
except for amount of starting material (Don't worry, the quantity of
resin/matrix/slurry {whatever you want to call it} can handle that amount
of cell lysate).

o  Pass the lysate/slurry through cartridge via syringe, wash, etc as
recommended.

o  Transfer cartridge into opening of 1.5 mL ufuge tube, quick spin at top
speed in ufuge (reach top speed then stop). You ought to have eluted
about another 50 to 200 uL wash ... cartridge is now dry enough for
elution step ....
o  Transfer cartridge into opening of fresh 1.5 mL ufuge tube already
containing 2 uL of 5 N NaOH (ANY NaOH stock, or any other basic pH
solution for that matter must always be stored in plastic, NEVER in GLASS
!!!!!!!) .... then transfer this cartridge/ufuge tube assembly into ufuge
rotor with cap open towards centre of rotor ... put 50 uL hot clean water into
cartridge orifice (hot water taken from, for example a 50 mL screw cap
polypropylene tube kept in 80oC water bath at least 15 minutes before
use). 

o  Quick ufuge again, remove/discard cartridge, close the ufuge tube,
vortex, heat at between 37oC and boiling for 15 to 5 min.

o  Quick spin tube again to pellet condensate. Add 6 uL of 3 M NaOAc, pH
4.5, then add 200 uL -20oC 100% ethanol, mix, spin, wash, dry, etc.

The alkaline conditions remove ALL RNA, which inhibits sequencing rx'ns, also
the amount of template generated (usually a few ug) should be sufficient
for MANY cycle sequencings, otherwise such a prep yields enough ds plasmid
for no more than three/four regular Sequenase type rx'ns (example, if you
want to sequence from both ends, from middle, etc).

regards

----------------------------------------------------------------------------
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
545 University Crescent                    home tel.: (204) 275-1204
Winnipeg, Manitoba                               FAX: (204) 945-8062
CANADA
R3T 5S6
------------------------------------------------------------------------------

In article <C5yH42.222 at ccu.umanitoba.ca> jrichmn at ccu.umanitoba.ca (Joy Richman) writes:
>I am using a GIBCO BRL cycle sequencing kit but have had intermittent
>success with sequencing, ie. no visible bands. I am pretty certain that it
>is a problem with my template since the control ds DNA works.  We are
>using the Magiprep miniprep kit from Promega to purify our plasmid DNA.
>Has anyone out there had a problem with the quality of plasmid DNA from
>this kit? Are there better ways of preparing the template? Is quantity of
>DNA essential to success? How much is the minimum that will work? All I
>know is that more than the recommended 50 fM is required!
>
>All suggestions will be gratefully received. 
>
>
>Joy Richman
>
>"The most important event in life is not birth or death but gastrulation."
>Lewis Wolpert
>jrichmn at ccu.umanitoba.ca
>
>
>
>-- 
>The most important event in life is not birth or death but gastrulation.
>Lewis Wolpert
>jrichmn at ccu.umanitoba.ca
>



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