Total RNA extraction...

kyungho1 at snu.ac.kr kyungho1 at snu.ac.kr
Mon Oct 9 23:25:43 EST 2000


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
degree
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...



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