TITLE: DNA/RNA extraction from fresh-frozen tissue, AllPrep DNA/RNA/miRNA Universal Kit
AUTHORS: Annika Fendler

[DESCRIPTION]
Protocol for combined RNA and DNA extraction from fresh-frozen tissue using the AllPrep DNA/RNA/miRNA Universal Kit.

[BEFORE_START]
Preparations:
FRN buffer: Add 42 ml Isoprop to new bottle
RPE buffer: Add 44 ml EtOH to new bottle
AW1 buffer: Add 25 ml EtOH to new bottle
AW2 buffer: Add 30 ml EtOH
DNAse I stocks: 550 µl RNase-free water to lyophilised DNAse I, aliquot and store at -20°C for 9 months)

Immediately
DNAse I: 70 µl RDD + 10 µl DNase I per sample
Proteinase K: 60 µl AW1 + 20 µl Proteinase K per sample

[STEPS]
SECTION: Tissue preparation
1. This protocol is for   
Optional: Weigh tissue

SECTION: Tissue preparation
2. Transfer tissue in 350 µL 600 µL  RLT + ß-ME in 2 ml DNA LoBind tube.

Make sure that the tissue does not defrost.
SECTION: Tissue preparation
3. Add a 5mm bead to each tube and lyse tissue in  TissueLyser for 2 min  @ 20 Hz
 


SECTION: Tissue preparation
4. Spin down for 1 min @ 9000 x g 

SECTION: Tissue preparation
5. Turn tube rack and lyse tissue for 2 min  @ 20 Hz

SECTION: RNA extraction
13. Incubate 10 min @ Room temperature 

SECTION: Tissue preparation
6. Spin down for 1 min  @ 9000 x g 

SECTION: Tissue preparation
7. Add lysed product to DNA Mini Spin Column
SECTION: RNA extraction
11. Add 50 µL 80 µL  Proteinase K, mix by pipetting

SECTION: RNA extraction
12. Add 200 µL  350 µL EtOH abs., mix by inverting, spin down liquid

SECTION: Tissue preparation
8. Spin 30 s @ max x g , repeat if any liquid remains on column

SECTION: Tissue preparation
9. Transfer column to a new collection tube and store tube at 4 °Cuntil DNA extraction

SECTION: Tissue preparation
10. Transfer flow-through to new 2 ml LoBind tube
SECTION: RNA extraction
14. Add 400 µL 750 µL EtOH abs. , mix by pipetting

SECTION: RNA extraction
15. Add 700 µL to RNeasy spin column and spin for 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
16. Repeat until all liquid passed through the column
SECTION: RNA extraction
17. Add 500 µL RPE

SECTION: RNA extraction
18. Spin 30 s  @ max x g  and discard flow-through

SECTION: RNA extraction
19. Add 80 µL DNase I working solution directly onto the membrane and incubate 15 min at Room temperature 

SECTION: RNA extraction
20. Add500 µL FRN

SECTION: RNA extraction
21. Spin 30 s  @ max x g and don't discard flow-through

SECTION: RNA extraction
22. Add flow-through again to column 
SECTION: RNA extraction
23. Spin 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
24. Add 500 µL RPE

SECTION: RNA extraction
25. Spin 30 s @ max x g  and discard flow-through

SECTION: RNA extraction
26. Add 500 µLEtOH abs

SECTION: RNA extraction
27. Spin 2 min @ max x g  and place column in new collection tube

SECTION: RNA extraction
28. Spin 2 min @ max x g  and place column in new 1.5 ml collection tube

SECTION: RNA extraction
29. Add 30 µLof RNase-free H2O

SECTION: RNA extraction
30. Incubate 1 min @ Room temperature 

SECTION: RNA extraction
31. Spin down1 min @ 8000 x g 

SECTION: RNA extraction
32. QC: Qubit    and tape station   

48. 
SECTION: DNA extraction
33. 350 µL AW1 auf DNA Mini Spin Column geben

SECTION: DNA extraction
34. Spin 30 s @ max x g  and discard flow-through

SECTION: DNA extraction
35. 80 µLProteinase K working solution directly onto membrane

SECTION: DNA extraction
36. Incubate 5 min @ Room temperature 

SECTION: DNA extraction
37. Add 350 µL  AW1

SECTION: DNA extraction
38. Spin 30 s @ max x g  and discard flow-through

SECTION: DNA extraction
39. Add 350 µL AW2

SECTION: DNA extraction
40. Spin 2 min  @ max x g  and place column in new 2 ml collection tube

SECTION: DNA extraction
41. Spin 1 min  @ max x g  and place column in new 1.5 ml collection tube

SECTION: DNA extraction
42. Add 50 µL    directly onto membrane

SECTION: DNA extraction
43. Incubate 1 min  @ Room temperature

SECTION: DNA extraction
44. Spin down 1 min  @ 8000 x g 

SECTION: DNA extraction
45. QC: Qubit DNA    and tape station