This Protocol Can Take Either Two Or Three Days, Depending on the Amount of RNA Desired

-LiCl RNA Prep

This protocol can take either two or three days, depending on the amount of RNA desired.

Before you start you will need (the amount of each needed is dependent of the size of you prep):

·  Extraction Buffer

0.1M LiCl

0.1M Tris

pH to 8 with HCl then add

10mM EDTA

1% SDS

Make up the volume with ddH20

·  CIA

24:1 Chloroform/pentanol

·  Phenol

100g phenol

0.1g 8-Hydroxyquinoline

44ml H20

·  4M LiCl

·  Also DEPC water, 70% ethanol, 100% ethanol and 3M NaAc (pH 5.2)

Day 1

Soak pestle and mortar in 0.1M NaOH for a couple of hours to get rid of residual RNase. Grind mycelium in the usual fashion and transfer to sterile oakridge tube containing an equal volume of extraction buffer and phenol (or eppendorf depending on size of prep). Mix by inverting tube for 1 min. Add 0.5 vol. CIA and invert repeatedly for 30 sec. It is imperitive to keep them on ice during the above procedure.

Spin for 30 min at 10K (4°C) and transfer upper phase to a fresh tube and add 1vol. of 4M LiCl, leave on ice in a cold room overnight.

Day 2

Spin down tubes for 20min. 10K at 4°C, decant the supernatant and wash pellet in 70% ethanol, re-suspend in DEPC water and transfer to eppendorf. Add an equal vol. of phenol:CIA and vortex for 30 sec, then spin at max for 10min at 4°C. Take the top phase. This can be repeated if lots of muck is present in interface. Add 2 vol. of 100% ethanol and 0.1 vol. 3M NaAc (pH 5.2) (You may wish to split the sample if you have too much RNA solution to enable 2 vol. ethanol & 0.1 vol. NaAc to fit in an eppendorf). Leave to precipitate overnight at -20 or -70. (it is possible to precipitate for at least an hour if pressed for time, but be reminded that the yield will decrease).

Day 3

Spin down for 20 min, 4°C at 10K. Remove supernatant and wash in 70% ethanol and air dry in speed-vac. Re-suspend in either TELS or DEPC water (depending on the application RNA is for), the volume will also be dependent on how concentrated you want your RNA to be.

Check RNA by gel electrophoresis or Spectrophotometry.

Typical approx yields for this prep are:

Starting in eppendorf (small prep): 50mg per tube total

Starting in oakridge (large prep) : 1.5mg per tube total