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S oluti on- bas ed lso lat i on
Tri Pure lso lati on Reagent
V. Protocol for preparing RNA, DNA and protein from animal tissue(basd on tlÉ method 0f Chomczynski, 7 993)
Nate: Fora düailed, step-by-step procedure and fortips on handling different typesof sample,see he package insertsuppliedwith therHgslt.
+,',' ;,,,, ..4dd,,ct¡lirob'irir. (0.2,rn.l pei l
'm.l T¡Füie).:.
.. aite.,ri=lgbr¡!¡¡ly.-1-$¡s;¡.fn!,u!.¡¡,.e¡2, -i1é..in,i1 L!,Xo,?,.5.1! ,
+
I
Y.' j,,,:,', - :, lsolarion ofnNA/gr¡tei f ::'';,
; ;1,,l .;,' ;
'w,':,ry cPb1eandorsanrcq¡.h
Iü,,t,?rgsipitatc,r¡rdth ethq.nQJ { E¡9.!J,},,..,,ÍA 3, m:á.&-o/q.EtO-H per l, nnl iFlíe),.',
M ix by inversionlncutute 2- 3 min at 15to 25"C
:-.j.!:, : ../. .a. .::.¿:.:.,.:j-:-
+' -r'eentrlft¡gs2000x !i 5 m.¡,n:at? to 8!C , . ,Separate pellet and phenol/EtOH supernatant
+Isolationof protein
Phenol/EtOH supernatant
+
IüPrecipitate w ith isopropa nol
(1 .5 ml per 1 ml TriPure)Mix by inversion
lncubate 10 min at 15to 25"C
Iv
Cenrifuge 12,000 x g,
10minat2to8'CDiscard supernatants*ers DEtrC':treilcd
lsú¡bl0-15mir d
!¡HrpÉÉclri.ü?S6itCf¡tl nl@Hp, ..: ...-!,,fril..,l.-riF.{,,9_,,-,,,.,,
It..:. r.:rlril :t::ir:iriir:lir)t.::ir:tr:t:,:r.. :,_i .
,,ri$s,qi,..&g{50sxr; r
.i:::r¡,5¡*t*rt z:tir'..,a:111,,[tüf¿rÍtssplmatant"i::':.,.::i,.,,,.. :':
fso|lionofpeinP henolEtoH supernatant
IvPrecipitate w ith isopropa nol
(1 .5 ml per 1 ml TriPure)Mix $r inversion
lncubate 10 m¡n at 15 to 25"C
++
Centrifuge 12,000 x g,10 min at 2 to 8'C
Discard supernatant
Itlet inResuspend pell..
0.3M GuHClin 95% EIOH(2 ml per 1ml TriPure)
lncubate pellet in wash 20min at 15 to 25'C
IvCentrituge 7500x g,5 min at 2 to 8"C
Discard supernatant
+Repeat wash step 2 x
+Wash protein pellet in
2 ml 100% EIOHVortex
lncubate 20 min at 15 to 25'CI
YCentrifuge at 7500 x g, 5 min at 2 to 8"C
Discard supernatant
IvDry pel let un der vacuum 5 - 10 m in
(orair dry)Add 1% SDSand incubate at 50"C to
disolve pelletSediment insoluble material 10,000 x g,
10minat2to8'CIt
Transfur su pernatant to new il be
Store probin at -1 5 to -25'C
s5"- 60"C'to.og