Water saturated phenol

C Coward cc122 at mole.bio.cam.ac.uk
Mon Apr 3 07:30:28 EST 2000


"P. Jeffrey Lewis" <JLEWIS at upei.ca> writes:

>Hi,

>We are preparing to do some RNA extraction from a Gram positive organism
>and have settled on a protocol that relies on GITC and Bead-based
>disruption.  In the protocol that we are modifying a second extraction

<snip>

You may be interested in this protocol that we have successfully used for
the extraction of RNA from the Gram-positive Lactococcus lactis. It relies
on the cell wall being weakened by lysozyme treatment but saves the
hassle we found with bead-beating methods. I don't have a reference for
the method I'm afraid but could possibly track it down if required:

Unless otherwise stated all centrifugations were at 13000rpm in a
microfuge

1) Take 10ml culture at OD 0.5-0.8 or 5ml OD 1.0

2) Spin 10' at 3K [can now snap-freeze in liquid nitrogen if required].

3) Resuspend in 1ml protoplasting buffer on ice
	50 mM Tris pH 7.4
	3 mM MgCl2
	25% sucrose
	5 mg/ml lysozyme

4) Incubate 10' at 4 degrees C

5) Pellet in eppendorf tubes for 1 min

6) resuspend in 50 ul RNA prep buffer I
	20 mM NaOAc pH 5.5
	1mM EDTA
	25% sucrose

7) Lyse cells with 200 ul RNA prep buffer II
	20 mM NaOAc pH 5.5
	1 mM EDTA
	0.5% SDS

8) Extract with 250 ul acid phenol
	phenol equilibrated with 20 mM NaOAc pH 5.5
        [we purchased this from PhiBio]

9) Incubate 5' at 60 degrees C with gentle shaking. Spin at 4 degrees C
for 5 min. Place briefly at 60 degrees C to clear cloudiness.

10) Remove aqueous phase

11) Re-extract as above for a total of 3 extractions.

12) Extract 1x w/ chloroform:IAA

13) Add 1/10 volumes NaOAc (pH 5.2) and 2.5 volumes of EtOH. Precipitate
overnight at -70 degrees C.

14) Spin pellet down at 4 degrees C for 30 minutes

15) Wash RNA in 70% EtOH and resuspend 100uL
DEPC-dH2O. Quantitate by taking OD260/280 of 5ul in 500ul

16) Store at -70 degrees C with 1/10 volumes NaOAc (pH 5.2) and 2.5
volumes EtOH. Precipitate and wash appropriate aliquots as required.

Chris




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