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tags: molecular biology (tutorial), ebook, DNA extraction
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# HotSHOT DNA Extraction
##### *version 1.0: 2023-06-08*

## Summary
This protocol is the first version of a simple method for isolation of PCR-ready genomic material named HotSHOT.
This method was developed to be used with mice tissue (see Truett et al., 2000), and is very well tested by many labs for subsequent PCR amplification due to its simplicity and for being a very robust DNA preparation method.
This protocol has been succesfully applied in experiments with insect samples, but can be tailored to work with any animal taxa of interest.

:::info
- It is suggested by original protocol to prepare alkaline lysis solution fresh each day, but some versions suggest each month.
- Neutralizing solution is stable at room temperature for long periods (months-years).-Tris-HCl is **NOT** Tris-base!
- The main cause of failure is too much tissue for the volume of alkaline lysis solution.
:::

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## Materials

### Equipment needed
1. Lab coats and gloves of different sizes
2. P1000, P200, P10 pipette and filtered pipette tips
3. If prepping specimens by pulling legs or equivalent, several forceps and ways to clean forceps after usage (use flame and ethanol)
4. Paper filter disks to blot samples – if samples were fixed in ethanol
5. 1.5 ml Eppendorf DNA LoBind tubes (Eppendorf: 022431021)
6. Conical 50ml tubes to prepare stocks
7. 0.2 ml strip thin-walled PCR tubes (Thermo Fisher Scientific: AB0451)
8. Standard thermocycler (e.g., Bio-Rad Laboratories, cat. no 1861096EDU)
9. Benchtop microcentrifuge (e.g., Thermo Fisher Scientific mySPIN 6, cat. no. 75004061)
10. Qubit instrument or equivalent to measure DNA concentration
11. Mixer/vortex (any rotator mixer/vortex with a gentle setup)
12. Beakers and Erlenmeyer flasks
13. Precision balance and weigh boats

## Procedure
1. Prepare **Lysis buffer** according to table below (double check molarity of solutions a priori and volumes!)

| Reagent | If using 1.5ml tube | If using conical 50ml tube |
| ------------------- | ------------------- |:--------------------------:|
| Nuclease-free water | 1200 μL | 24 mL
0.1 M NaOH solution| 400 μL | 8 mL
0.5 M EDTA | 0.64 μL | 12.8 μL
**Total** | ~1600 μL| ~32 mL

2. Prepare **Neutralization buffer** according to table below (double check molarity of solutions a priori andvolumes!)

| Reagent | If using 1.5ml tube | If using conical 50ml tube |
| ------------------- | ------------------- |:--------------------------:|
| Nuclease-free water | 1 mL | 40 mL
40 mM Tris-HCl | 0.64 μL | 12.8 μL
**Total** | ~1 mL | ~40 mL

:::warning
:warning:
You can adapt the reactions for your needs. Measuring 6 mg of Tris-HCl might be tricky, so perhaps favor making stock solution in conical tube and create small aliquots when conducting experiments. Amounts don’t need to be super precise (630 mg and 0.25 g should work fine).
:::

3. Remove pieces of tissue from your specimens (it can be a leg of an ant, whole specimen if using small phorid flies, mammal fur, tip of bird feathers, etc.), blot sample (if sample was stored in ethanol or dissection was performer in any particular media) and put sample in PCR tubes.

:::warning
:warning:
The liquid volume should be sufficient to allow for complete submersion of the tissue.
:::

4. Label tubes accordingly – I recommend creating a spreadsheet in your lab notebook where vial numbers = collection code or any other specimen code you can match to your vials. In this way you can use simple numbers to label your experiment (i.e., 1 to infinity).

5. Turn on thermocycler. Create following protocol (times change in different versions I found online, I amusing the following based on protocol provided by a lab studying phorid flies): 65°C for 18 minutes, 98°C for 2 minutes, hold at 4°C ∞. Lid temperature can be left at 80-95°C.

6. Add 15 μL of lysis buffer to each tube. Quick vortex and centrifuge.

7. Move vials to thermocycler and start incubation. Wait until samples are cooled to 4°C.

8. Add 15μL neutralizing buffer to each sample, mix well by vortexing.

9. If plan is to conduct future genomic extraction on specimens, move them back to original tube and fill with ethanol. Keep HotSHOT solution in PCR vials, storing at 4°C (up to 3 months) or -20°C for long-term use.

10. If plan is to get rid of tissue (i.e., if only using legs or any other tissue), centrifuge at max speed for a coupleof minutes to pellet the debris and use supernatant for PCR reactions.

11. Proceed to PCR reactions.