Equipment-
Direct-zol kit (label columns N=one per sample)- in 1.52 on Pedro’s shelf
DNase 1 and buffer defrosted shortly before use- in -20ᵒ freezer in 1.06
Ethanol (95-100%)- under fume hood
Autoclaved/RNase-free eppendorf tubes (labelled N= two per sample + several for DNase 1 digestion)
eppendorf holder x2
p1000 pipette and filtered tips
p200 pipette and filtered tips
p10 pipette and filtered tips
RNase-free spray
Steps proceeding homogenisation of recently defrosted flies in trizol reagent.
1.
Alcohol then RNase-free equipment, bench, fumehood, centrifuge buttons
2.
Firstly, prepare DNase 1 reaction mix in appropriate no. tubes to make enough for all samples. Multiply each volume below by the no. of samples. Add reagents in following order-
• 5µl DNase 1 (reconstituted at 1U/µl- add 275µl RNase free H₂O per vial)
• 8µl 10xDNase 1 reaction buffer
• 3µl RNase free H₂O
• 64µl RNA wash buffer (diluted 1/5 with 95-100% ethanol- 12ml W.B./48ml eth)
Mix well and leave reaction mix to one side on ice

3.
Centrifuge sample eppendorf tubes at 12 000 xg for 1 minute at 4ᵒ (or longer if necessary)
4.
Transfer supernant into into an eppendorf tube
5.
Add one volume ethanol (95-100%) directly to one volume sample homogenate in trizol (1:1)
6.
Mix well by vortexing
7.
Load mixture from eppendorf into column. Place column into collection tube and centrifuge at 10-16 000 xg for 1 minute at 4ᵒ
8.
Transfer column into a new collection tube and discard collection tube containing flow through
9.
Add 400µl RNA wash buffer to wash column
10.
Centrifuge column in collection tube at 10-16 000 xg for 30 sec at 4ᵒ, then discard flowthrough
11.
Add 80µl of the DNase 1 reaction mix directly to the column matrix and leave to incubate for 15 minutes at room temp
12.
At end of incubation, centrifuge at 10-16 000 xg for 30 sec at 4ᵒ
13.
Add 400µl RNA pre-wash (diluted 1/5 with 95-100% ethanol- 10ml RNA prewash/40ml eth)
14.
Centrifuge columns at 10-16 000 xg for 1 min at 4ᵒ, then discard flow-through
15.
Repeat steps 13 & 14
16.
Add 700µl RNA wash buffer (diluted 1/5 with 95-100% ethanol) to the column
17.
Centrifuge at 10-16 000 xg for 1 min at 4ᵒ, then discard flow-through
18.
To ensure complete removal of wash buffer, centrifuge the columns for an additional
2 mins in the emptied collection tube
19.
Carefully, transfer the the column into an eppendorf tube
20.
Add 50µl of RNase-free H₂O directly to the column matrix (for max elution repeat
20&21 or increase H₂O volume/ for highly conc RNA use >25µl H₂O)
21.
Centrifuge at 10-16 000 xg for 1 min at 4ᵒ
22.
Store eluted RNA at -80ᵒ (>-70ᵒ)