Sequencing with Promega DNA

Andre Hamel hamel at ccu.umanitoba.ca
Tue Apr 20 10:26:52 EST 1993


We've had consistent results using 5- 10 mL overnight culture worth of
cells, alk.SDS lysis/2.5 M NH4OAC neutralization, spin 10 min 4oC, etc as
per Magic mini prep kit instructions, except 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, then
remove cartridge, close cap, boil for 5 min, then add 6 uL of 3 M KOAC, pH
4.5 plus 200 uL -20oC 100% ethanol, mix, sit -70oC for no more than 10
min, ufuge top speed for 10 min, decant/discard supernate, add 1 mL -20oC
80% ethanol, close cap, invert few times, ufuge top speed for 5 min,
drain, dry .. for Sequenase, dissolve pellet in 10uL freshly premade
priming mix (buffer and about 3- 12 ng primer) by vortexing
occassionally over a few minutes, then boil 5 min in 100 mL beaker of water,
slow cool in about to about 30oC, then place on ice, quick ufuge (to top
speed for a second), add labelling mix as recommended by Sequenase kit,
keeping all on ice, incubate 15oC for 2 min, back on ice. Before labelling
rx'n (whilst primer is annealing for instance) prelabel and aliquot into
four seperate capped tubes 4.5 uL of each dideoxy mix. Then once labelling is
started, place these dd mix tubes into PREHEATED 42oC heat block/water
bath. After labelling rx'ns (2 min, NO MORE), aliquot 3.5 uL of label rx'n
into each preheated dd mix tube mixing by drawing up anddown few times
(of course using fresh upipet tip for each dd mix), incubate 42oC for 2- 3
min, then stop by adding 4 uL of stop/tracking/loading dye solution (99%
formamide/bromophenol blue/xylene cyanole 1 mM EDTA, pH 8), heat to no
less than 70oC (90oC okay) prior to loading. For longer sequences keep
times same, simply increase labelling dNTP mix conc.

cheers

In article <baxevani-190493172040 at 130.14.22.89> baxevani at ncbi.nlm.nih.gov (Andy) writes:
>Has anyone had problems sequencing DNA purified using Promega's magic
>column
>miniprep kits? My results have been quite variable since I've started using
>it,
>with some samples sequencing beautifully and others not showing up on the
>autorad at all -- I haven't experienced this kind of variability in the
>past.
>Of particular interest would be if any of you incorporate additional steps
>prior to starting the sequencing reactions. I'm using USB's Sequenase to
>run
>the reactions.
>
>Thanks in advance.



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