single_fly_DNA_extraction_DiNV.md

Single Fly DNA Extraction Protocol in Single Tubes for DiNV Containing Flies

The general steps of a DNA extraction are:

1. Homogenization - break open hard tissue to access cells (exoskeleton)
2. Cell lysis - break open cells to access nuclei, mitochondria, and the genome
3. Protein/RNA/debris removal - removal of all other cellular components while leaving DNA in solution
4. DNA purification - separating out DNA from the extraction solution and eulting it in a solution suitable for downstream use

Read this for more information on steps of DNA extraction, what they mean, and what methods are out there for extraction. This protocol is an alcohol precipitation based method.

This protocol uses a Puregene DNA Purification Kit from Qiagen for all reagents and methods, except for RNase A which is purchased separately from Qiagen.

Other materials:

• 70% ethanol (100% ethanol and molecular grade water)
• 100% isopropanol
• 1.5mL centrifuge tubes
• Kimwipes
• Ice bucket and ice
• Autoclave-sterilized homogenization pestles
• p200, p20, and p2 pipettes and filter tips

Note that this protocol is slightly different than the general single fly extraction protocol because of the presence of virus in your samples. Contamination of viral DNA is easy and needs to be avoided. You must include extraction controls, extract your samples in a specific order, and use filter tips.

Users should start out with single flies in individual 1.5mL tubes that have been frozen at -20C since collection.

Aliquots of kit solutions can be made if many users are using the kit as to avoid contamination. Pour or pipette yourself 15-50mL aliquots of cell lysis solution, protein precipitation solution, and DNA hydration solution. Keep these labeled and sterile.

Use sterile technique and avoid contamination of samples throughout this process.

Planning how to extract samples

• With most lab processes where you have to process a lot of samples in batches, you would randomize the order samples get prepared in. However, because of the concern of contamination of viral DNA, your samples have to be ordered in a very specific order for this type of DNA extraction
• If you have a small number of samples (typically less than 48), you can do them all in one day of extraction. You should order your samples will all of the control flies first, and all of your virus flies second
• If you have more flies to do than in one day of extraction, split up your controls and virus infected flies into groups based on how many rounds of extractions you will do. You should order the samples you will be extracting on each day with controls first, then virus infected samples
• Additionally, for every day you do an extraction, you should include two extraction controls. These are empty tubes that get treated like they have a fly in them, and get the whole process done to them. This can help you identify contamination. Make one extraction control tube your first tube you extract, and the other one should be the last tube you extract
• For example, you should have a set of samples that looks like this:

tube	treatment
extraction control 1	NA
1	control
2	control
3	control
4	control
5	control
6	infection
7	infection
8	infection
9	infection
10	infection
extraction control 2	NA

Extraction Steps

Note that filter tips are used for all pipetting!!

Homogenization

1. Chill the cell lysis solution on ice until it becomes cloudy. This may take ~30 minutes
2. Make sure the heat blocks are turned on to 65C and 37C (or the incubator oven is at 65C if you are doing more than 24 samples - note that you have to set the incubator oven to ~71C to get it to be 65C)
3. Keep fly samples on ice
4. While on ice, add 100ul of cell lysis solution to each fly tube
5. Homogenize each fly with a sterile pestle by hand. Squish and mash up the fly, being careful not to splash the liquid. Each fly gets a new pestle, and pestles should be bleached and autoclaved before using
6. Keep pestles until you are finished and clean then afterwards
7. Use the mini-centrifuge to briefly spin down the homogenized fly tubes

Lysis

8. Incubate tubes in the heat block/oven for 15 minutes at 65 degrees C
9. While tubes are incubating, make a dilution of RNase A to 1mg/mL. Stock lab solutions may be at 100mg/mL or 10mg/mL. Only make about what you need. Do not store diluted RNase A for further use because it is no longer in its storage buffer
10. After incubation, let tubes cool to room temp on the bench
11. Add 2ul of the diluted RNase A to each tube
12. Invert tubes to mix 25 times by flipping the tube rack over and over, this way all tubes get mixed the same number of times
13. Use the mini-centrifuge to briefly spin down the tubes after mixing
14. Incubate tubes at 37 degrees C for 40 minutes in either the heat block or the incubator oven
15. While this is incubating, you can prepare the final tubes. Make 1 new 1.5L tube for each sample and add 100ul of fresh 100% isopropanol to each tube. Then label the tubes: on the lid label with the sample number/ID and "DNA", on the side, repeat the sample number/ID, DNA, the date, and your initials
16. Additionally while the samples are incubating, make fresh 70% ethanol by mixing 35mL of 100% ethanol with 15mL molecular grade water for a 50mL conical, or 10.5mL of 100% ethanol with 4.5mL of molecular grade water for a 15mL conical
17. Cool tubes to room temperature on the bench

Protein and debris removal

18. Add 33ul of protein precipitation solution to each tube
19. Vortex tubes for 10 seconds each
20. Place tubes on ice for 5 minutes
21. Centrifuge tubes at ~14,000rpm for 3 minutes (maximum speed in small centrifuges). If you have more than 24 samples this has to be done in 2 batches

DNA precipitation and purification

22. After centrifugation, transfer the supernatant to the final tubes with isopropanol in them. Be very careful to not pipette up any debris in the pellet. The volume will be close to 130ul
23. Invert tubes gently 50 times to mix by flipping the tube rack over and over
24. Centrifuge tubes ~14,000rpm for 5 minutes, making sure to put all tubes in the centrifuge with the hinge of the caps facing out of the centrifuge. This way the "back" of the tube is where the pelleted DNA gets pushed to
25. Check all tubes for a DNA pellet - this may not be visible, but you should still "act" like a pellet is there because sometimes the DNA is not enough to see
26. Discard supernatant by pouring the liquid off away from the pellet gently. This waste is not hazardous and can be rinsed down the sink after the process, it is easiest to pour into a cup and rinse later
27. Add 100ul freshly prepared 70% ethanol to each tube
28. Invert tubes ~2 times to mix
29. Centrifuge tubes ~14,000rpm 1 minute, again with the hinge of the cap facing out of the centrifuge
30. Discard supernatant by pouring the liquid off away from the pellet gently
31. Let tubes dry on the benchtop by inverting on a kim wipe for 30 minutes to 1 hour
32. Resuspend pellets in 20ul DNA hydration solution (or other desired volume. The less volume you use the more concentrated the DNA will be)
33. Let DNA resuspend overnight on the benchtop
34. The next day, freeze DNA at -20C if not using right away
35. DNA can be used to check concentration (Qubit) or directly into a PCR