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POLY A+ RNA PREPARATION using proteinase K and SDS

NOTE: All solutions should be autoclaved or treated with 0.1% DEPC. Homogenizer probe should be treated with 0.5M NaOH for about 5 min, then rinsed with sterile H20 and STE. Econo column should be washed with 0.5M NaOH then several aliquots sterile H20.

1. Wash cells with PBS.

2. Resuspend thoroughly at 107/ml in STE (0.1M, NaCl, 10mM Tris 7.4, 1mM EDTA), predigested proteinase K to 200ug/ml and SDS to 0.5% and homogenise with an ultraturax. For mouse tissues (one brain or 10-15 E13 embryos) homogenise in 10ml STE with 1mg/ml proteinase K, 0.5%SDS in a 50ml tube and then top up to 40ml with STE, 0.5% SDS (PK final 200ug/ml).

3. Incubate 60íC 1h then 37íC 20'. Make up to 0.5M with respect to NaCl and for tissues spin 3.5K 20íC for 10min. Transfer with a pipette to a fresh tube.

4. Add oligo dT-cellulose suspension (typically 0.25ml packed volume) and place on wheel for 1 hour. [To prepare oligo-dT ad to a 10ml tube, wash in 0.1M NaOH and spin down 2' in bench top 3K. Wash twice in binding buffer (0.5M NaCl, 10mM Tris 7.4, 1mM EDTA, 0.1% SDS) and make to a 50% slurry and add to lysate. Resuspend for each sample as the oligodT settles fast in a pipette.]

5. Spin down oligo-dT in lysate and wash twice with 15ml binding buffer. Transfer to alkali-treated (i.e. RNASE-free) econo-column.

6. Wash with at least 10ml of binding buffer. Wash with 2ml wash buffer (0.1M NaCl, 10mM Tris 7.4, 1mM EDTA, 0.1% SDS).

7. Elute RNA with 2 x 0.4ml of elution buffer (10mM Tris pH 7.4, 0.1 mM EDTA) into an ependorf tube. Vortex and remove 50ul for OD. Split sample into two ependorf tubes and add 1.0ml ethanol and35ul 5M NaCl. Freeze at -20íC.

This method can also be used for solid tissues. There is no hard rule for how much material to use, but since even a small piece of tissue is equivalent to a large number of cells, it is advisable to use a large volume of STE/proteinase K/SDS. The procedure works well for adult brain, mouse embyros and cell lines but not very well for other adult tissues. Acid phenol is the best technique for the latter.

100ml (400ml)

H2OTris 2MNaCl 5MEDTA 0.25MSDS 10%

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