Help! RNA extraction
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.
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
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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