TITLE: Qiagen AllPrep 96 DNA/RNA protocol for bee abdomens (FFAR)
AUTHORS: Jocelynz  

[DESCRIPTION]
Qiagen AllPrep 96 DNA/RNA protocol for bee abdomens (FFAR)

[STEPS]
SECTION: Before beginning protocol
1.   must be added tobbefore use.  Add 20 µL20 Molarity (M)    per 1 mL  . Stock solution of   should be prepared fresh or frozen in single use aliquots.   containing   can be stored at room temperature for up to 1 month.

 

 

 

 

SECTION: Prepare samples
2. UV sterilize supplies for 2 96 well plates: stainless steel beads, 8 reagent reservoirs, 2 collection microtube plate (light blue) and strip caps (clear), 2 brand new s-blocks, 4 more s blocks, 4 elution microtube plates (dark blue) and strip caps (white), and 96 well microplates (for aliquots). Label foil lids for aliquots. Turn on water bath and set to 70 °C 

SECTION: Prepare samples
3. Add 1 stainless steel bead to each well of collection microtube plate
SECTION: Prepare samples
4. Working on dry ice to prevent samples from thawing, add 1 bee abdomen to each collection microtube. Enter each sample ID into your plate map as you work and keep used vials stored in order.
SECTION: Prepare samples
5. Remove from dry ice and immediately add 
 


SECTION: Prepare samples
5.1. For larger bee abdomens such as melissodes, add 500 µL   

SECTION: Prepare samples
5.2. For smaller bee abdomens such as lasioglossum, add 350 µL   

SECTION: Prepare samples
6. Seal with strip caps and place in tissue lyser, placing a folded kim wipe between strip cap tops and adapter to ensure a tight seal
 

 

SECTION: Prepare samples
7. Lyse at 25 Hz for 2 min 
 

SECTION: Prepare samples
8. Remove plates from lyser and adapters, rotate plates 180 degrees, and place back in tissue lyser
SECTION: Prepare samples
9. Lyse at 25 Hz for 2 min 
 

SECTION: Prepare samples
10. Centrifuge 3486 x g, 7 min, 20-25 °C 

 

SECTION: Prepare samples
11. Open a brand new s-block and place DNA filter plate on top

 


SECTION: Prepare samples
12. Transfer 350 µL supernatant to DNA filter plate and seal with AirPore tape

SECTION: Prepare samples
13. Centrifuge 3478 x g, 7 min, 20-25 °C . Check that all liquid has flowed through columns, repeating centrifugation if needed

 



SECTION: Prepare samples
14. Place DNA filter plate on new s-block and put aside at room temperature to 4 °C for later use
 


SECTION: RNA purification
15. Place RNA filter plate on s-block
SECTION: RNA purification
16. Add 350 µL    to s-block containing flowthrough from step 13. Pipette up and down 3 times to mix.

SECTION: RNA purification
17. Transfer samples to RNA filter plate. Seal with AirPore tape
SECTION: RNA purification
18. Centrifuge 3486 x g, 7 min, 20-25 °C , discard flowthrough

SECTION: RNA purification
19. Add 800 µL   and seal with AirPore tape

SECTION: RNA purification
20. Centrifuge 3486 x g, 7 min, 20-25 °C , discard flowthrough

SECTION: RNA purification
21. Add 800 µL   and seal

SECTION: RNA purification
22. Centrifuge 3486 x g, 16 min, 20-25 °C , discard flowthrough

SECTION: RNA purification
23. Place RNA filter plate on top of elution microtube plate
SECTION: RNA purification
24. Add 60 µL RNase-free water directly to center of each filter membrane and seal

 

SECTION: RNA purification
25. Incubate at room temperature 1 min 

SECTION: RNA purification
26. Centrifuge 3486 x g, 7 min, 20-25 °C 

SECTION: RNA purification
27. Repeat steps 23-26, eluting into same microtube plate
SECTION: RNA purification
28. Distribute product into 10 µL aliquots and seal with labeled foil lids. Seal remaining product in elution microtube plates with white strip caps

SECTION: RNA purification
29. Store at -20 °C - -80 °C 

SECTION: DNA purification
30. Place   in 70 °C water bath
 

SECTION: DNA purification
31. Get out your DNA filter plate and s-block from step 14
SECTION: DNA purification
32. Add 800 µL   and seal with AirPore tape

SECTION: DNA purification
33. Centrifuge 3486 x g, 7 min , discard flowthrough

SECTION: DNA purification
34. Add 800 µL   and seal

SECTION: DNA purification
35. Centrifuge 3486 x g, 16 min , discard flowthrough

SECTION: DNA purification
36. Place DNA filter plate on top of new elution microtube plate
SECTION: DNA purification
37. Add 50-100 µL preheated   directly to the center of each filter membrane and seal

SECTION: DNA purification
38. Incubate at room temperature for 5 min 

SECTION: DNA purification
39. Centrifuge 3486 x g, 7 min 

SECTION: DNA purification
40. Repeat steps 36-39, eluting into same plate
SECTION: DNA purification
41. Distribute product into 10 µL aliquots and seal with labeled foil lids. Seal remaining product in elution microtube plates with white strip caps

SECTION: DNA purification
42. Store at -20 °C - -80 °C