Soil and litter I followed the protocol from the extraction kit.
I used the salt extraction protocol of Aljnabi & Martinez (1997).
1. Prepare insect trap samples: Pour out ethanol and let dry for a while. Most of the ethanol needs to be removed, but insects may remain wet. This helps to submerge them into the buffer in step 2.
2. Pipette 15 ml of extraction buffer (0.4 M NaCl, 10 mM Tris-HCl pH, 2 Mm EDTA pH 8.0 (and 2 % SDS) and 1,5 ml of 20 % SDS (sodium dodecyl sulphate) on top of the sample in labelled Falcon tube.
3. Add 10–30 µl of Proteinase K digestive enzyme into the tube. Vortex.
- The amount of protK depends of the total biomass of the sample: more insects, more protK (I used 20 µl in general).
4. Leave to digest at + 60 ºC for overnight in the orbital shaker
5. Transfer ~15 ml of clear lysis solution into a new tube. Avoid transferring insects along the solution. Small amounts if insects don’t affect the extraction.
6. Add ~11,25 ml of 6 M NaCl to the tube containing lysis solution. Vortex for 30 seconds.
7. Place the tube into a centrifuge and spin at 10 600 rpm for 30 minutes.
8. Take 25 ml of supernatant from the tube and place into a clean labelled 50 ml Falcon tube.
- These are the final tubes that the DNA will be stored in and should thus be labelled with sample ID, date, your own name
- If the salt + other unwanted have not spun down to the bottom of the tube after 30 minutes making it difficult to cleanly pipette the supernatant, you can transfer e.g. 30 ml of the supernatant + whatever comes along to a fresh tube (not labelled so detailed) and centrifuge it again for 5-10 mins, then transfer 25 ml to a labelled tube
9. Add 25 ml of isopropanol to each tube. Mix by turning tube upside down a few times.
10.Place tube into -20 ºC for one hour.
- One hour is minimum time, can be kept in freezer for longer, e.g. overnight
11.Centrifuge tubes at +4 ºC and 13 200 rpm for 20 minutes.
12.Pour out isopropanol and wipe tubes.
13.Add ~2 ml of ice cold 70 % EtOH. DNA pellet should me submerged.
14.Centrifuge tubes at +4 ºC and 13 200 rpm for 8 minutes.
15.Pour out EtOH and leave tubes to dry at +60 ºC for one hour (or at room temperature overnight).
16.Add 50–1000 µl of sterile H2O to each tube and leave at room temperature for 1-2 hours.
17.Store at -20 ºC.