TITLE: Historical Samples Extraction
AUTHORS: Dakota Betz

[DESCRIPTION]
Extraction protocol for historical samples. Requires overnight incubation and rotary mixer.

[GUIDELINES]
Important Safety Note: Complete extraction steps in a fume hood and be very careful about any skin contact. Extraction buffer contains hazardous chemicals with inhalation hazards. Separate hazardous lab waste is needed for all waste products. See Safety Warnings below for more information.

[STEPS]
SECTION: Extraction Buffer Preparation
2. In a 50 mL conical:
50 g guanidine isothiocyanate
5.3 mL 1M Tris-HCl pH 7.5
5.5 mL 0.2M EDTA
10.6 mL 20% Sarkosyl
1 mL B-mercaptoethanol
Add 50 mL of ddH2O.
 


SECTION: Day 1  - Incubation
5. For each sample: place tissue in a 1.5 mL eppendorf tube with 200 uL of extraction buffer.
SECTION: Day 1  - Incubation
6. Incubate on hot plate at 55 °C   .

SECTION: Day 2 - Silica Bead Preparation
7. 
 


Pipette or vortex G-Biosciences Silica Magnetic Beads thoroughly to re-suspend.
SECTION: Day 2 - Silica Bead Preparation
8. Transfer 20 µL of beads into a clean, labelled 1.5 mL eppendorf tube.
Note: This volume is scaled (amount for a single sample: 20uL). 
 

If scaled to 8 samples: Use 160 uL of beads.

SECTION: Day 2 - Silica Bead Preparation
9. Place the tube on the magnetic stand for 30 -  60  seconds until clear: 30 s - 1 min . 
Use a pipette to discard the supernatant.

SECTION: Day 2 - Silica Bead Preparation
10. Remove the tube from the magnetic stand. Add 100 µL  ddH20 or Elution Buffer (do not use Elution Buffer for museum samples).
Resuspend the beads by pipetting or vortexing.
Note: This volume is scaled (amount for a single sample: 100uL)
 

If scaled to 8 samples: Use 800 uL ddH20.
SECTION: Day 2 - Silica Bead Preparation
11. Place the tube on the magnetic stand for 30 -  60  seconds until clear: 30 s - 1 min . 
Use a pipette to discard the supernatant.
SECTION: Day 2 - Silica Bead Preparation
12. Repeat steps  10-11 twice:   

SECTION: Day 2 - Silica Bead Preparation
13. Remove tube from the magnetic stand. Add 20 µL  ddH20.
Note: This volume is scaled (amount for a single sample: 20uL).

If scaled to 8 samples: Use 160 uL ddH20.
SECTION: Day 2 - Extraction
15. Reminder: Complete extraction steps that involve lysate in fume hood.

If any solid debris remains, centrifuge extraction 2 minutes: 12000 rpm, 2 min .

SECTION: Day 2 - Extraction
16. Add extraction lysate (liquid only, should be ~200uL) to each tube with 20uL of silica beads mixture. Discard any debris left over from centrifugation.
SECTION: Day 2 - Extraction
17. Add 200 uL of 100% Ethanol to lysate + beads.  Gently mix and incubate in a rotary mixer for 15 minutes: 15 min .

SECTION: Day 2 - Extraction
18. Place tubes on magnetic stand for approximately 5 minutes, until clear:  5 min . 
Use a pipette to discard the supernatant.

SECTION: Day 2 - Extraction
19. Pipette 200 uL of PE Buffer (Qiagen) and incubate in a rotary mixer for 10 minutes: 10 min .

SECTION: Day 2 - Extraction
20. Repeat steps 18 - 19 twice:   . When ready to place on magnet stand for the final time (moving on to step 21), spin tubes down briefly (mini centrifuge) to get beads out of cap.
SECTION: Day 2 - Extraction
21. Keeping tubes on the magnetic stand, allow beads to air dry for 20 - 45 minutes:  30 min  - 45 min .

SECTION: Day 2 - Extraction
22. Remove tubes from the magnet and elute beads in  20-50 uL of ddH2O. Use pipette to gently mix and use the elution H2O to encourage beads off the side of the tube. Incubate at 55 °C for 10 minutes: 10 min at 55 °C .

SECTION: Day 2 - Extraction
23. If needed, briefly spin tubes down (mini centrifuge) to get liquid out of caps.
Place tubes on magnetic stand. Wait until liquid is clear.
Use a pipette to move supernatant to a clean/new labeled tube, and discard the beads.
SECTION: Day 2 - Silica Bead Preparation
14. Pipette to resuspend. If applicable, aliquot  bead mixture into number of tubes equal to number of samples you are extracting (number you scaled this protocol by). Each should have ~ 26.3uL if aliquotting from larger volume.

If scaled to 8 samples:  Should have 8 tubes with ~20 uL of bead mixture in each.

SECTION: Safety Note
1. Complete extraction steps in a fume hood and be very careful about any  skin  contact. Extraction buffer contains hazardous chemicals. Separate hazardous lab waste is needed for all waste products. See Guidelines & Warnings for more information.
SECTION: Extraction Buffer Preparation
3. ALTERNATIVE Buffer Prep SCALED for Fewer Samples:

Volumes below are scaled to number of samples  but you must make batches for at least 10--choose a total volume that is easy to check by eye for addition of ddH2O (amount for 1 sample: 0.2g guanidine isothiocyanate, 21.2uL 1M Tris-HCl, 22uL 0.2M EDTA, 42.4uL Sarkosyl, 4uL B-mercaptoethanol; bring volume up to 200uL).

0.2 g guanidine isothiocyanate (for 10 samples: 2g)
21.2 µL 1M Tris-HCl pH 7.5 (for 10: 212uL)
22 µL 0.2M EDTA or 8.8 µL 0.5M EDTA (for 10: 220uL)
42.4 µL 20% Sarkosyl (for 10: 424uL)
4 µL B-mercaptoethanol (for 10: 40uL)
Add 200 µL  ddH2O (for 10: 2mL)


SECTION: Day 1  - Incubation
4. Important Safety Note: Buffer mixture is flammable. Use caution if sanitizing with flame between specimens.