TITLE: SOP - Lysis C plate based DNA extraction
AUTHORS: Petra Korlević, Mara Lawniczak

[DESCRIPTION]
This custom minimally morphologically destructive DNA extraction protocol was developed for malaria transmitting Anopheles mosquitoes as a cheap and fast alternative to kit-based approaches. DNA extracted using this method can be used unpurified but diluted for singleplex or multiplex PCR. It can also go unpurified straight into Covaris shearing followed by Illumina library preparation or, if quantification or quality is uncertain, it can be purified and used for whole genome sequencing. It is important to note that the times, temperatures, and volumes suggested here were primarily adapted for use with Anopheles mosquitoes, but these are quite flexible and can be adapted for different Diptera species (such as a 5-fold increase in volume for larger species like Glossina) and probably beyond. 

Our SOP is derived from two publications in which lysis buffer C was singled out as the best buffer for both present-day plate-based mosquito DNA extractions (least salt carryover into PCR) and historic museum single-tube DNA extractions (minimally morphologically destructive). 

For more information please refer to the original publications:

Makunin, A., Korlević, P., Park, N., Goodwin, S., Waterhouse, R. M., von Wyschetzki, K., Jacob, C. G., Davies, R., Kwiatkowski, D., St Laurent, B., Ayala, D., & Lawniczak, M. K. N. (2022). A targeted amplicon sequencing panel to simultaneously identify mosquito species and Plasmodium presence across the entire Anopheles genus. Molecular Ecology Resources, 22(1), 28–44. 10.1111/1755-0998.13436
Korlević, P., McAlister, E., Mayho, M., Makunin, A., Flicek, P., & Lawniczak, M. K. N. (2021). A Minimally Morphologically Destructive Approach for DNA Retrieval and Whole-Genome Shotgun Sequencing of Pinned Historic Dipteran Vector Species. Genome Biology and Evolution, 13(10). 10.1093/gbe/evab226

[BEFORE_START]
Prepare the appropriate amount of lysis C buffer and add Tween-20 and Proteinase K just before use. 

[STEPS]
SECTION: Day 1 (buffer preparation and sample incubation)
1. Prepare an adequate volume of lysis buffer C in a 15 mL or 50 mL falcon tube using the following table (make sure to adjust the initial concentration of stock components if they differ):
 
      ComponentStock or LOT No.Initial conc.Final conc.Final volume (single 100 μL reaction)Final volume(multiply with No. of samples)Water72.95 μLTris pH81 M200 mM20 μLEDTA pH80.5 M25 mM5 μLTween-20100%0.05%0.05 μLProteinase K20 mg/ml0.4 mg/ml2 μL

 
 
SECTION: Day 2 (DNA extract and specimen plate storage)
3. 
SECTION: Day 1 (buffer preparation and sample incubation)
2. Pour the prepared and well mixed lysis buffer C into a 50 mL reservoir for multichannel pipette dispensing or keep in the falcon tube for single pipette dispensing.

NOTE 1: Add Proteinase K just before use for maximum enzymatic activity.
NOTE 2: A larger volume of lysis buffer C without Proteinase K can be stored at room temperature for a prolonged time; just take an adequate aliquot and add Proteinase K to it once you are ready to perform the overnight extraction.
NOTE 3: Out of precaution we advise to keep track of stock and LOT numbers of all components in case you notice any contamination or efficiency issues down the line.
NOTE 4: For Anopheles mosquitoes we found that 60 µLto 100 µL per well is an ideal volume in order to submerge the whole specimen and where the DNA concentration is still optimal in a 1:10 dilution for subsequent PCR reactions.
NOTE 5: Always include at least one extraction negative control per plate (well with only buffer).


SECTION: Day 2 (DNA extract and specimen plate storage)
2. 
SECTION: Day 1 (buffer preparation and sample incubation)
3. If insects are stored in ethanol: 
SECTION: Day 1 (buffer preparation and sample incubation)
4. If insects are dried (e.g. pinned or from silica gel tubes):
SECTION: Day 1 (buffer preparation and sample incubation)
3.1. Remove as much ethanol from the plate using pipettes while avoiding causing any damage to the insects.
SECTION: Day 1 (buffer preparation and sample incubation)
3.2. OPTIONAL: Leave the open plate in the incubator at 37 °C until all ethanol evaporates (typically takes about 15 min if there is < 10 μL of liquid remaining in each well). 

SECTION: Day 1 (buffer preparation and sample incubation)
3.3. Add an appropriate volume of lysis buffer C (this depends on the size of insect) to each well once ethanol has fully evaporated, making sure the samples are submerged.
SECTION: Day 1 (buffer preparation and sample incubation)
4.1. Dispense an appropriate volume of lysis buffer C into each well of a plate. Keep any columns or plates containing aliquoted lysis buffer C loosely closed with strip caps to avoid bits of insects falling in as you are plating them. 
SECTION: Day 1 (buffer preparation and sample incubation)
4.2. Using tweezers take the individual insects and dip in lysis buffer C (leave any appendages behind in the original tube), clean tweezers thoroughly in between with ethanol and Kimtech wipes or Azo wipes. 

NOTE 6: Dry insects tend to be extremely statically charged, and due to all our plasticware and consumables being equally charged, body parts often detach and fall onto the benchtop or even into plate wells. One thing we saw helped with this issue is to rehydrate the desiccated tissues before submerging in lysis buffer. This can be done by placing the dried samples into a styrofoam box filled with wet paper towels, and incubating in an oven at 37 °C for 120 min to 180 min prior to handling.

SECTION: Day 1 (buffer preparation and sample incubation)
5. Overnight incubation:
SECTION: Day 1 (buffer preparation and sample incubation)
5.1. Once all insects are submerged in appropriate lysis buffer volumes, close the plate with strip caps making sure no caps are loose as that might cause evaporation.
SECTION: Day 1 (buffer preparation and sample incubation)
5.2. If there are substantial droplets visible on the walls of the wells, do a short spin down in a plate centrifuge. 
SECTION: Day 1 (buffer preparation and sample incubation)
5.3. Incubate the plate 15 min in an oven at 56 °C. Shorter periods of extraction also work well and can cause less morphological damage, so the exact volumes and times for this protocol could be modified depending on the insect species and size of material being extracted.

SECTION: Day 2 (DNA extract and specimen plate storage)
6. After incubation is complete, transfer the lysis buffer C (now referred to as “DNA extract”) to a fresh 96-well plate. For long term storage keep this plate sealed with PCR grade foil to avoid evaporation, typically at 4 °C but freezing is also possible depending on the desired downstream application. 

SECTION: Day 2 (DNA extract and specimen plate storage)
7. For downstream amplifications using PCR (e.g. ANOSPP), prepare a 1:10 dilution of the DNA extract with PCR grade water. This can be used both as a DNA template in PCR reactions and for PicoGreen quantification, as the unpurified lysate is too dirty (dyes, salts) to be QC’d accurately without this dilution. In contrast, for whole genome sequencing using Illumina kits, you can start with the unpurified lysate as a template for Covaris shearing without prior purification.
SECTION: Day 2 (DNA extract and specimen plate storage)
8. For the now extracted insect specimens, add 100 µL-150 µL of 70-100% ethanol, seal the plates with strip caps and store in the fridge.

NOTE 7: The volume will depend on the size of the insect, while the concentration will depend on what the sample will be used for in the future (70% for better morphological feature preservation, 100% for leftover DNA preservation)