Horseshoe Crab DNA Extraction with Zymo Quick DNA Miniprep Plus kit - Protocol Only

Process samples following steps from previous Extraction Post with a few minor changes:\

• Increased PBS from 100ul - 300ul solution
• Increased Proteinase K from 15 - 20ul
• Used more tissue
• 2 hour thermomixer time

Tissue Processing

1. Take samples out of -80 freezer to thaw slowly in ice bucket
2. Label 1 1.5mL tube for each sample with “Sample # PBS” and 1 1.5mL tube for each sample with “Sample #”
3. Add 270ul of DI type II water to each of the PBS tubes
4. Add 30ul of PBS solution to each of the PBS tubes Above 2 steps create 300ul of 1X PBS solution
5. Add 300ul of DNA/RNA shield to each of the 1.5mL tubes labeled with “Sample #”
6. Prepare scalpel handle and forceps by cleaning with 10% bleach solution, DI II water, and 70% ethanol solution From this step forward this cleaning process will simply be referred to as “cleaning protocol”
7. Place new piece of tinfoil on benchtop and put new scalpel blade on handle
8. Remove first sample from tube
9. Carefully cut a small piece of tissue away from exoskeleton
10. Finely chop piece of tissue into thin transparent layer
11. Place pea sized amount of tissue into tube labeled with corresponding sample number and “PBS”
12. Place remainder of tissue back into original collection tube
13. Place remainder of sample back in -80 freezer
14. Let sample soak for 5-10 minutes while prepping other samples
15. Remove used scalpel blade from handle
16. Repeat steps 6 through 15 for remaining samples
17. After soaking, move each sample to corresponding tube of DNA/RNA shield
18. Get proteinase K from -20 freezer and put in ice bucket - currently open tube lives in box with samples that have been processed
19. Get blue tissue buffer out of Zymo Quick DNA Miniprep Plus kit
20. Add 150ul of blue tissue buffer to each of the sample tubes
21. Add 20ul of proteinase K to each sample tube
22. Vortex and centrifuge each sample on countertop machines for ~10 seconds
23. Set thermomixer to 55 C
24. Once warm, put samples in thermomixer for 2 hours at at 1200 rmp

Extraction

1. While samples are in thermomixer, set up new 1.5mL tubes with “Sample #” (one for each sample)
2. Turn off thermomixer
3. Remove samples from thermomixer and centrifuge at 13000rcf for 1 minute
4. Carefully remove supernatant from above pellet in each tube and deposit liquid only into new tube
5. Add enough HCL Tris to 1.5ul tube and labeled “Tris HCL” and place in thermomixer on 70 degrees Each sample will need 100ul of Tris HCL so calculate correctly
6. Set up spin columns for each sample -yellow tubes- in clear collection tubes -no cap- and label with “Sample #”
7. Get “liquid waste” container
8. Add 450ul of Genomic Binding Buffer from Zymo kit to each of the tubes containing samples
9. Vortex and centrifuge each sample for ~5 seconds
10. Add 700ul of each sample to corresponding spin column set up
11. Centrifuge tubes at 13000rcf for 1 minute
12. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
13. Remove remaining liquid from sample tubes and place in corresponding spin column setup
14. Centrifuge tubes at 13000rcf for 1 minute
15. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
16. Move yellow columns to new collection tubes and discard used ones with tips
17. Add 400ul DNA pre-wash from Zymo kit to each collection tube setup Labelled DNA pre-was with “Pre” on top to avoid confusion
18. Centrifuge tubes at 13000rcf for 1 minute
19. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
20. Add 700ul of DNA Wash Buffer to each spin column
21. Centrifuge tubes at 13000rcf for 1 minute
22. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
23. Add 200ul of DNA Wash Buffer to each spin column
24. Centrifuge tubes at 13000rcf for 1 minute
25. Pour liquid that collected in bottom tube into liquid waste and plac yellow spin column back in corresponding tube
26. Make new 1.5mL tubes labelled with “sample number, Date, horseshoe crab, DNA, NJA”
27. Move yellow spin columns to labelled tubes created in last step
28. Discard remaining liquid waste from collection tubes and discard collection tubes in tips
29. Add 50ul of warmed 10mM Tris HCL to each tube to incubate for 5 minutes Carefully drip liquid directly onto white disc in yellow tube
30. After incubation, place samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spin at max speed for 1 minute
31. Remove samples from centrifuge and add 50ul of warmed 10mM Tris HCL to each tube
32. Let samples incubate for 5 minutes
33. After incubation, place samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spin at max speed for 1 minute
34. Remove samples from centrifuge and discard yellow spin columns in tips
35. Set up freezer box with name, date, lab name, DNA extraction, and box number or use one that isn’t full
36. Label new tubes from a set of strip tubes with sample numbers
37. Remove 8ul from each 1.5mL samples and place in corresponding strip tube
38. Place samples and strip tubes in Box in -20 upright freezer on Puritz shelf