totalRNA extraction from infected protoplast

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HELISA
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totalRNA extraction from infected protoplast

Post by HELISA » Fri May 12, 2006 5:46 pm

Hi Guys,
I’m working in virology lab and from time to time I have to struggle with total RNA isolation from protoplasts infected with virus.
Unfortunately, my RNA is very often slightly degradated, even if I am using typical procedure to avoid this effect i.e. DEPC treated H20 is used for preparation of all buffers, I’m wearing gloves, am not sneezing and crying close to my samples, however there is sometimes great temptation to do that 
So, I am asking you for some advices how to avoid degradation of RNA?
I have to specify the procedure which I am using:
1) Protoplast RNA isolation buffer:
50 mM Tris-HCl (pH 7.5)
100 mM NaCl
1 mM EDTA
1% SDS
0.3 mg/ml bentonite
2) Twice phenol/chloroform extraction pH 4.2
3) Precipitation with LiCl 8M pH5.2; 2h -70 C
4) Precipitation with NaoAC pH5.3 in ethanol; over night -70C
I know that the clue of the degradation is somewhere in extraction procedure, because separation on denaturizing gel is O.K. (checked by positive control, viral RNA).
Any ideas?
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seaseasea
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Post by seaseasea » Tue May 16, 2006 4:20 am

do you have a special space for RNA operation?
do you use RNAase-free EP tubes?
do you use respirator?
carefully,i think you must be successful.

hope this helps.
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