Sequencing with Promega DNA

Andre Hamel hamel at ccu.umanitoba.ca
Tue Apr 20 17:24:49 EST 1993




Yes, I use ALL of the DNA from a 5- 10 mL overnight culture.

Sorry for the lack of clarity (Hemingway I ain't), I'll try again;

o The cleared lysate is mixed with a few mL of slurry supplied with the kit,
o then passed through a cartridge via the use of a 3 mL syringe attached
  to cartridge at the Leur lock end 
o then washed similarly with the supplied washing solution via same syringe
o after no more liquid can be removed from cartridge then the cartridge is
  removed from syringe.

The Promega Magic mini prep cartidge is then fitted snuggly into the
opening of standard 1.5 mL ufuge tubes, with the Leur lock end on top.

A quick spin at top speed, then cartridge (now dried inside) is inserted
into a fresh 1.5 mL ufuge tube that already contains 2 uL of 5 N NaOH.
This is then placed into ufuge rotor with cap towards centre of
rotor. Finally, 50 uL of hot water (at least 70oC) is placed into orifice,
then is quick spun (reach top speed then stop). Remove catridge from tube.
Close cap. Quick vortex, then boil sample for 5 min, then add salt, 4x vol
etoh, etc.

Hope this is more understandable. Works EVERY time for us.

regards

Andre

In article <9304201647.AA11333 at net.bio.net> RGB12955%USA.decnet at USAV01.GLAXO.COM ("USA::RGB12955") writes:
>Andre,
>
>I always enjoy your postings, but I may have missed something here.
>
>Do you use the entire miniprep (5-10ml) for one set of reactions?  Also,
>could you expand on this section of your posting:
>
>...after the washing the mini column/ cartridge elute the DNA with 50 uL hot
>water (90oC will do), which fits into the top end of the cartridge whilst it's
>in the ufuge in a 1.5 mL ufuge tube containing 2 uL of 5 MN NaOH, with cap still
>attached placed into centre of rotor .. quick spin to top speed for only a
>second...
>
>I got kind of confused.
>
>Thanks
>



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