Qiagen DNeasy extraction

(using Qiagen blood and tissue kit)

Protocol adapted from Lotterhos lab protocol, DNeasy® Blood & Tissue Kit (Quick-Start Protocol April 2016), & DNeasy Blood & Tissue Handbook

Tissue Lysing

Need:

scale
sterile forcepts (store in 50 ml tube with 10% bleach then rinse with DI water spray bottle over waste container OR spray with 70% EtoH and use a flame to burn off any residual EtoH and tissue.)
scalpel or razer (clean between samples with same steps as forcepts)
weighting boats
1 ml tubes
ice container
Qiagen blood and tissue kit
- Buffer ATL (if there are precipitates heat at 56 C and mix gently)
- Proteinase K

Steps:

1. preheat thermomixer to 56 °C
2. clean area with 10% bleach, water, then 70% ethanol
3. prep ice container to store tissue samples while working
4. remove tissue samples from freezer and let thaw; once thawed place on ic
5. while waiting for samples to thaw
- 5.1. label 1 ml tubes
- 5.2. add 180 µl of Buffer ATL & 20 µl of Proteinase K into labeled 1 ml tubes
6. tare weighing boat on scale
7. carefully remove tissue from storage tube with forcepts and use sterile blade to cut a piece of tissue roughly 20mg in weight
8. plase tissue on weighing boat and measure weight (should weigh around 20mg +/-2mg, if not remove or add tissue as nessesary)
9. once correct weight, place tissue in prepped 1 ml tube
10. record weight in lab notebook
11. seal tube and store on ice until all samples are complete
12. clean instruments between each tissue sample by soaking in 10% bleach, spraying with DI water (holding over waste container/glassware), then wiping down with kimwipe. (could also sterilize with DI water, 70% ethanol, then then running instrument through an ethanol lamp filled with 100% ethanol)
13. repeat steps 5 through 11 for every sample
14. confirm that all tubes are sealed, then vortex for 15 seconds
15. place samples on thermo-mixer set at 56 °C at 600 rpm (check after 2 mins to make sure everything is sealed)
16. samples can be left for at least 2 hours or overnight to allow for all tissue to be completely lysed

DNA extraction

Need:

96-100% molecular grade ethanol
Qiagen blood and tissue kit
- DNeasy spin columns
- DNeasy collection tubes
- Buffer AL
- Buffer AW1
- Buffer AW2
1.5 or 2 ml microcentrifuge tubes (for final storage)
warmed 1X low TE buffer (37ºC); may need to make by diluting 20X low TE buffer

Steps:

1. prep and clean bench with 10% bleach, water, then 70% ethanol
2. label spin-column tubes and final storage tubes
3. remove samples from thermo-mixer
4. add 200 μl Buffer AL to samples, then vortex
5. add 200 μl Ethanol (96-100% molecular grade) to samples, then vortex
- Note: A working solution of Buffer AL & ethanol can be made to be aliquoted into sample tubes.
6. pipet the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube
7. centrifuge for 1 min at ≥ 6000 x g (8000 rpm)
8. change gloves after each centrifuge step
9. discard the flow-through & collection tube
10. tranfer spin column to a new collection tube
11. pipet 500 μl Buffer AW1 into the spin column
12. centrifuge for 1 min at ≥6000 x g (8000 rpm)
13. discard the flow-through & collection tube
14. tranfer spin column to a new collection tube
15. pipet 500 μl Buffer AW2 into the spin column
16. centrifuge for 3 min at 20,000 x g (14,000 rpm)
17. discard flow through, place back into collection tube, & spin again for 1 more min
18. make sure all the spin column membranes are dry after this step; spin again for 1 min if not
19. discard the flow-through & collection tube
20. tranfer spin column to a new 1.5 ml or 2 ml microcentrifuge tube (for final storage)
21. elute the DNA
- 21.1. if only eluting once:
  - add 100 μl of warm low TE buffer (warm in thermomixer, 37ºC) to the center of the spin column membrane
  - incubate for 1 min at room temperature (15–25°C) then centrifuge for 1 min at ≥6000 x g (8000 rpm)
- 21.2. if eluting twice (to increase yeild):
  - add 100 μl of warm low TE buffer (warm in thermomixer, 37ºC) to the center of the spin column membrane
  - incubate for 1 min at room temperature (15–25°C) then centrifuge for 1 min at ≥6000 x g (8000 rpm)
  - transfer spin column to a new 1.5 ml or 2 ml microcentrifuge tube (label tube differently than the first to note that it has the second elution in it)
  - add another 100 μl warm low TE buffer
  - incubate for 1 min at room temperature (15–25°C)
  - centrifuge for 1 min at ≥6000 x g (8000 rpm)
22. store in fridge for a few days or freeze at -20 or -80 if not using immediately

Possible Next Steps

Qubit
PCR then gel electrophoresis