NH4Ac-Miniprep: The Protocol

* * at alf1.ngate.uni-regensburg.de
Fri Aug 30 08:55:43 EST 1996


Hi..
Two days ago I posted a question why my NH4AC-Miniprep doesn't work
after I have the solutions stored for a month.
The most reasonable explanation came from Dr. Hiranya S. Roychowdhury
saying that an NH4Ac-solution is not stable for a long period at room
temperature. I still wonder what happens to it and if it helps to
store it at 4'C.
Other people mentioned the NaOH-neutralisation due to atmospheric CO2.
Well I used to make fresh lysis buffer before but found that it
actually is fairlly stable if you close the bottle tightly. You can
also check it's quality during the prep very easily. (Cell suspension
becomes clear and when you open the tube you see "strings" of DNA from
tube to lid)

Someone else asked me for reference and protocol.
Sorry... I don't know where it was published but here's the procedure:

- spin down 1.5mL of ON culture
- discard supernatant and resuspend cells in 200uL bufferA 
  (Qiagen bufferP1 works as well)
- add 400uL of bufferB, shake slightly (no vortex) and incubate on ice

  for 5 min
- add 300uL of bufferC, vortex and incubate on ice for 10 min
- spin for 15 min (prepare new caps filled with 500uL Isopropanol)
- pour supernatand into new caps with Isoprop. and shake once
- spin for 15 min
- discard supernatant and wash pellet (almost invisible) with
  1mL EtOH Abs.
- dry pellets and resuspend in 50uL H2O
  (use 5uL for digestion)

BufferA:
	Tris-HCl pH 8.0	50mM
	EDTA		10mM
	RNase A	100 ug/ml

BufferB:
	NaOH		200mM
	SDS		1%

BufferC:
	Ammoniumacetate	7.5M


Have fun...
Mike






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