Help! RNA extraction

Unknown Unknown
Mon Mar 27 06:28:37 EST 1995


In article <3ksca9$7v7 at hippo.shef.ac.uk>, Andrew Brooks <A.Brooks at shef.ac.uk> says:
>
>Does anybody have a really good RNA extraction method for small amounts of tissue.
>Preferably one that can all be done in a 1.5ml microfuge tube.


HI
We have used the following method for a number of years; with good results.
We can extract  good yields (suitable for RT-PCR)  from 1million spleen or lymph cells

Lysis Buffer:
	5ml	 4M Guandinium Thiocyanate
	125ul	 1M Sodium Citrate
	45ul	 2- Mercaptoethanol
	250ul	 10% Sarcosyl

All procedures are carried out on Ice.

Add 500ul of Lysis Buffer to tissue sample.
Disrupt  tissue by passing up and down a  22g needle and syringe.  Vortex 30s.
Add 50ul 2M Sodium Acetate. Vortex 30s.
Add 500ul water-saturated Acid-Phenol. Vortex 30s.
Add 100ul Chloroform. Vortex 30s.

Incubate for 15m. on ice. Vortex every 5m.

Spin down on mircocentrifuge (13000rpm)

Careful remove aqueous(top) phase 2 x 200ul to a fresh tube to this add 400ul IsoPropanol

Freeze  at -20 for 1hour or store samples at this stage.

Spin sample at 13000rpm
Wash in 800ul 80% Ethanol x 2

Dry under vacuum.

Resuspend in DEPC treated water.

Hope you find this protocol useful for your work

B-Butler at nimr.mrc.ac.uk





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