Monday, November 10, 2003

Description
Seed RNA Extraction Protocol

Procedure
After harvesting, siliques should be immediately frozen in liquid N2. Samples are stored at -80C until the extraction
Place all solutions, eppen etc on ice to cool. Set centrifuge to 4C. All steps are performed on ice and all centrifuge steps are at 4C and 10,000 rpm
All supernatant removal should also be carried out with a fine pippette. We use either a 200 ml yellow pippette tip attached to a pasteur pippette for this, or a fine pasteur pippette made using a bunsen burner

Add 300l of EB and 300 ml of PCI to an eppen tube on ice

Cool the mortar & pestle with liquid N2, add 10 seed pods (or 0.05g of 8DAF siliques) per sample and homogenise very well, adding N2 as necessary to keep sample frozen

Once fully ground, use small spoon to transfer sample to the eppen tube with the buffer

vortex for 3 min and divide the sample into 2 eppen tubes

Centrifuge for 1 min and collect sup into new eppen

Add an equal volume of PCI and vortex

Centrifuge for 5 min and collect sup into new eppen

Centrifuge again for 5 min and collect sup into new eppen

Add 0.1 vol 3M NaOAc and 3 vol 100% EtOH, invert 4 times to mix

Place at -80C (dry ice) for 10 min

Centrifuge for 5 min, discard sup and vacuum dry pellet

Dissolve pellet with 100l ddH2O (may take time)

Centrifuge for 10 min and collect sup into new eppen

Add equal volume of 4M LiCl, invert 4 times to mix

Place on ice on 4C cold room overnight or -80C (dry ice) for 1 hr

NEXT DAY

Centrifuge for 15 min, discard sup. Small pellet should be visible, be careful as it moves easily

Add 1 mL of 2M LiCl and carefully invert once to mix

Centrifuge for 2 min and discard sup

Add 1 mL of 70% EtOH and invert once to wash

Centrifuge for 2 min, discard sup and vacuum dry

Dissolve pellet in 5 to 10l ddH2O

Combine the 2 solutions/sample into one eppen

Centrifuge for 5 min and collect sup into new tube

Store at -30C or lower

Expected RNA amounts: 4DAF, 1-2g; 6DAF, 2-3g; 8DAF, 6-8g; 10DAF, 8-10g; 12DAF, 6-8g; 14DAF, 3-4g



Recipes
Extraction Buffer (EB)
1M Tris-HCl pH9.0
1% SDS
autoclave
PCI
Phenol:Chloroform:Isoamyl alcohol (25:24:1)
The 24:1 chloroform:isoamyl alcohol can be prepared in advance and stored. PCI should be prepared fresh by mixing 1 part of TE-saturated phenol to 1 part of the 24:1 chloroform:isoamyl alcohol solution. This can be stored up to 1 week at room temp in the dark.
Also prepare:
3M NaOAc pH5.6 (autoclaved)

4M LiCl (autoclaved)

2M LiCl (autoclaved)

100% and 70% EtOH

mortar and pestles

13 eppen tubes/sample

ddH2O (autoclaved)



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