Login

Join for Free!
118863 members


totalRNA extraction from infected protoplast

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

totalRNA extraction from infected protoplast

Postby 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?
Image
Image
HELISA
Death Adder
Death Adder
 
Posts: 67
Joined: Sat Feb 25, 2006 12:55 am
Location: Illinois

Postby 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.
Happiness is my favourite!God is also my favourite!
Happiness is also God!
seaseasea
Garter
Garter
 
Posts: 20
Joined: Tue May 16, 2006 2:49 am
Location: China


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 0 guests