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RNA prep/Northerns


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#1 jodonogh

jodonogh

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Posted 25 April 2002 - 07:25 AM

Hi all-
Hope someone can help me! I want to start doing some Northerns on frozen mouse tissues - can anyone advise
me on the best RNA prep method for use to get good quality RNA for Northerns??
Any info on RNA preps/Northerns is gratefully received!

Thanks!
Jean


#2 philippe Meilleur

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Posted 26 April 2002 - 12:01 PM

Dear Jean, I hope this protocol will help...

1.Defreeze your samples.
2.Take 0,1g of tissu, put it in 1ml Trizol
3.Homogenization with ultra-turax (kind of blender) 3 times for 20 sec.
4.keep on ice
5.5 min. room temp.
6.+0,2 ml of chloroform
7.shake gently (15 sec.)
8.centrifugation 12 000rpm, 15min., 4 Celsius
9.Put surnatant in an other eppendorf
10.+0,5 ml Isopropanol
11.shake gently
12.room temp for 10 min.
13.centrifugation 12 000rpm, 15 min, 4 Celsius
14. remove surnatant, keep pelet and rince with 1ml of EtOH 70%
15.Vortex
16.Centrifugation 7 500rpm, 5 min., 4 Celsius
17.Dry the pelet
18. keep it at -80 Celsius or quantify your RNA (total)
You got your RNA!!

More informations: philippemeilleur@hotmail.com

Philippe Meilleur, Montreal, Canada.





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