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COCOA RNA EXTRACTION PROTOCOL

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					                 CACAO RNA EXTRACTION PROTOCOL


Extraction buffer was DEPC-treated and autoclaved except the Tris-HCL which was
prepared in DEPC-treated water.

Extraction buffer:
        2% CTAB
        100 mM Tris-HCL (pH 8)
        25 mM EDTA
        2 M NaCl
        0.05% spermidine trihydrochloride

       2% PVP (molecular weight 10,000) - Added just before use
       2% b-mercaptoethanol – Added just before use

Chloroform-isoamyalcohol (24:1)

10 M LiCl (DEPC treated before autoclaving or make up with previously treated DEPC
water)

100% ethanol

1% DEPC water – Diethyl pyrocarbonate is added to RO, filter purified water. The
container is vigorously shaken until it is thoroughly mixed, incubated at 37 0 C overnight
and then autoclave for 30-45 min.

Preparations:
1. Metal spatulas are foil wrapped and baked at 200 0C for 4 hours; These can also be
sprayed with Eliminase and rinsed with DEPC treated water.
2. 40ml and 30 ml oakridge tubes are filled with 1% DEPC water, allowed to incubate
overnight at 370C and autoclaved for 45 min. The water is discarded before use.
3. 38.5 ml polyallomer centrifuge tubes can be sprayed with Eliminase and rinsed at least
twice with DEPC water
4. Glass pipettes for use with the chloroform/isoamyl alcohol are baked at 200 0C for 4
hours.

PROTOCOL

1. To 15 ml of the premade extraction buffer in a 40 ml oakridge tube, add 2%PVP and
2% b-mercaptoethanol. Mix well and warm to 65 0C in a water bath.
2. Grind 0.5-0.7 g of leaf tissue in liquid nitrogen in a previously frozen mortar.
3. Transfer the finely ground powder to 15 ml of preheated buffer and blend for
approximately 1 minute in a Tissuemizer or Polytron.
The Tissuemizer or Polytron probe must be treated with Eliminase by placing 40 ml in a
tube and blending with the probe and repeating with 3 rinses of DEPC water. The probe
is treated likewise between each sample.
4. Place the tube into a 65 0C for 10 minutes.
5. Chill on ice for 1 min.
6. Add an equal volume (15 ml) of chloroform/IAA and shake vigorously.
7. Centrifuge for 10min at 10,000 X g (8500 rpm) at 4 0C.
8. Transfer the very viscous top layer to a new 30-40 ml oakridge tube and re-extract
with an equal volume of chloroform/IAA. Centrifuge as above.
9. Collect the top layer very slowly while carefully trying to avoid taking up the
chloroform or flocculent material at the interphase and transfer this to a 38.5 ml
centrifuge tube.
10. Centrifuge the supernatant at 15,000 rpm (31,000 X g) for 30 min at 4 0C to pellet any
contaminants and transfer the supernatant to a clean centrifuge tube. Discard the
insoluble material.
This step is very important for removing contaminating polysaccharides before
precipitating the RNA.
11. Add 0.25 vol of 10 M LiCl to the supernatant, mix well by pipetting and store at 4 0C
on ice overnight.
12. Centrifuge the sample for 30 min at 15,000 rpm (31,000 X g) at 4 0C to recover the
RNA.
13. Completely pipette out the supernatant and discard. Wash the pellet with cold 75%
ethanol 3 times to remove the remaining contaminants and air dry for 10 min. I add 5 ml
of cold 75% ethanol, mix and then centrifuge for 10 minutes at 10,000 rpm. Decant and
rinse twice with 2 ml of the cold 75% ethanol.
14. Dissolve the RNA in 100 ul of DEPC treated water and place in a microfuge tube.
(Gently pipetting the RNA solution up and down assists in resuspension.) Add an
additional 50 ul of DEPC treated water to collect any residual RNA from the tube. Keep
on ice while working. If the suspension is cloudy, centrifuge at 10,000 g for 5 min at 4 0C
and recover the supernatant. Store at –800C for future use.

To quantify:

Make a dilution of your sample in 10mM Tris. Make sure to set the dilution factor on the
spectrophotometer to what you have used and verify that the machine is set for RNA with
a factor of 40.
To check visually, prepare a .8 % agarose gel made with DEPC water, add ethidium
bromide and pour into a mold that was treated with DEPC water. Use TAE made with
DEPC water as the running buffer. Load 200-400 ng of sample onto the gel. Run at 75 –
100 volts for 30-45 min and view under UV.

Additional purification:

We have also used the RNeasy Min Protocol for RNA Cleanup by Qiagen to further
purify the sample.
To concentrate the sample, add 1/10 volume of 3M Sodium Acetate and 2.5 volumes of
cold 100% ethanol and place in a –200C for 20 min to overnight.
Spin at 40C for 15 min and decant the ethanol
Wash with 1 ml of cold 75% ethanol and spin for 5 min at 4 0C
Decant and air dry for 10 min.
Ressuspended in appropriate amount of water.

				
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posted:3/12/2010
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