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.

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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

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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