Total RNA extraction...
klevitsky at biochem.wisc.edu
Fri Oct 13 11:41:57 EST 2000
RNase contamination at any step would be devastating to the RNA. You must
make sure all glassware, tubes, and anything else coming in contact with
the RNA is RF, even the water in your reagents. Everything must be DEPC
treated and autoclaved. There are also reagents called RNase Away that
inactivate RNase without the need to incubate for prolonged periods of
time, like with DEPC. I had some trouble isolating total RNA from rabbit
hearts untill I got really anal about everything in the procedure and
RNase. Please forgive if this advice is too obvious, as it will be to
anyone experienced with RNA work. I am not certain what your level of
experience is. Good luck.
kyungho1 at snu.ac.kr wrote:
> Hello. everyone...
> I have been doing Total RNA extraction out of bovine muscle and fat
> tissues. I couldn't get pure
> band size...I tried several different methods to get accurate Total RNA
> using TRIzol regent and
> Guanidium method. I still didn't get it yet...
> When I got samples from cattle, I put samples into 50ml falcon tube.
> Then tissues directly in nitrogen tank.
> Trizol regent 10ml per musscle 1g.
> Homogenizing with electric homogenizer(IKA homogenizer)
> transfer samples into 1.7ml tubes. 1ml/tube
> add 0.2ml chloroform -> centrifuge 12,000g 15 min. 4 cel degree
> transfer aquueous phaese to new tube, add 0.5 ml isopropyl alcohol ->
> 12,000g 10 min. 4 cel
> remove supernate. add 1ml 75% ethanol -> vortexing -> 7,500g 5 min. 4
> cel degree
> remove supernate. vacum dry 5 min.
> dissolving the RNA with DEPC water 80ul.
> actually I followed the same procedure from Life technologies
> Electrophoresis.(6.5% formaldehyde composition)
> 1% agarose gel.
> DEPC 58.0 ml
> heating -> cooling to 50-60 cel degree
> add 10X MOPS 8 ml
> add Formaldehyde 14.0 ml -> cooling
> Formamide 5 ul
> 10X MOPS 2 ul
> Formaldehyde 1.8 ul
> EtBr 1 ul
> add Sample 9.2 ul -> 65 cel degree heating for 15 min. -> ice 3 min
> add loading buffer 1 ul.
> loading into gel... 60 voltage about 2 hr
> That is all my protocols for my experiment.
> Do you have any suggestion about my protocol.
> I really need someone's help and advice. I had been stop this process
> for 2 months.
> Hope to hear from you...
> Have a wonderful day...
> Sent via Deja.com http://www.deja.com/
> Before you buy.
Biochemistry Graduate Student
Peter J. Belshaw's Group
University of Wisconsin - Madison
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