HELP-RNA FROM PINE NEEDLES?!
hamel at ccu.umanitoba.ca
Thu Feb 18 19:03:14 EST 1993
In article <1993Feb15.124618.13924 at waikato.ac.nz> parkes at waikato.ac.nz writes:
>I'm a masters student doing molecular biology at Waikato.NZ and have been
>trying to make a cDNA library from Pinus radiata needles for what seems years
>but has been several months, one of several I'm ment to make for the project of
>finding a leaf specific promotor.
>Does ANYBODY know a method or alteration that may allow me to get a resonable
>conc. of RNA for the library? I've only got low yeilds of undegraded of more
>often degraded RNA.
>Any help/advice/protocol would be most welcome.
Have you referred to the Current Protocols in Mol Bio? In it are some very
good methods for plant RNA ext'n. From our experience, the CTAB based
methods work very nicely. Also, refer to Chomczynski and Sacchi 1987 Anal.
Biochem., 162:156-159 for a good guanidine thiocyante/acid phenol based
method... however for plants extensive chloroform (and/or ether)
extractions might be needed in addition to what is published (even with
the CTAB based methods, carbohydrates and phenolics can be nothersome
contaminants, thus chloroform and ether ext'ns become routine). Also
Chomczynski wrote an article appearing in the methods section of a 1992
issue of Nucleic Acids Res. regarding stability of RNA and using
formamide as the solvent of choice for dissolving the RNA after
purification (on ice of course). Also, formamide is good for eluting mRNA
from oligo dT purification, just alcohol ppt. with approp. salt and 3 - 4 x
vol 100% ethanol, mix, sit at -20oC overnight, spin, wash pellet in -20oC
80% ethanol, dry, dissolve in formamide. Don't apply total RNA to oligo dT
in presence of formamide because polyA and oligodT won't anneal very well,
for binding to oligodT, dissolve RNA TE-SDS buffer (10 mM Tris, pH 7.5/10
mM EDTA/0.5% SDS), (have RNA conc. at no more than 2 mg/mL), boil for 5
min., quick cool (in dry ice/ethanol or -70oC freezer packs/n-propanol),
mixing occassionally until ice JUST starts to form, then sit (float) tube
in crushed wet ice. Then add KCl to final conc. of 0.5 Molar, mix then
apply to oligo dT. If using oligo dT cellulose column it must have been
washed (including the oligo dT cellulose) with several volumes 0.1 N NaOH
(by completely resuspending up and down with pipet, better yet, mix in 50
mL plastic test tube (screw cap type) then rinsed with several volume
changes with 0.1% SDS in DEPC sterile filtered distilled water). The NaOH
ought to be filtered with glass fiber filters, ... remember NEVER store NaOH
solutions in glass, always in plastic. NaOH sol'ns can (and perhaps ought
to be) made in glass (because of heat generated, plastic may melt). But
NEVER store in glass due to production of sodium silicates which can be
bothersome as well as throwing off the hydroxide concentration.
If you need more details feel free to email me at
hamel at ccu.umanitoba.ca
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