Currently i have been using this protocol but result are not showing and if it shows, the DNA is very degraded:
1. Made 10ml Homogenization buffer.
50mM Tri/ Cl pH 8.3 - ul from 1M stock(500ul)
150mM Nacl - ul from 5M stock (300ul)
10mM EDTA - ul from 0.5M stock(200ul)
2. Cut 50mg fish tissue (Caudal Peduncle (100% ethanol preserved) mice it and i mix it with 500ul HB, add 0.1%(10ul) gently mix. later i add phenol (250ul) and chloroform(250ul) gently mix it and i centrifuge 10000 rpm, 5min, 4°C. i remove the aqueous phase to a new Eppendorf tube.
3. In the new tube with aqueous i add phenol (250ul) and chloroform(250ul) gently mix it and i centrifuge 10000 rpm, 5min, 4°C.
4. I remove the aqueous phase to a new Eppendorf tube. i add chloroform(250ul) gently mix it and i centrifuge 10000 rpm, 5min, 4°C.
5. I remove the aqueous phase to a new Eppendorf tube measure the volume with a pipette and i add 0.1 Na-acetate(5M) mix it gently and add 2,5 volume of 100 ethanol
After this step nothing is showing in the eppendorf tube, its just clear.
please any adjustment or recommedation of of Protocols i can use.. will gratly appreciate
- Badges
- Science topic