TITLE: ONT Post-PCR Pooling &amp; Purification for Fungal Barcoding
AUTHORS: Stephen     Douglas Russell

[DESCRIPTION]
Overview: The goals of this protocol are to pool your PCR product into a single 1.5 mL tube and to purify that product using magnetic beads.

Time required: ~45 minutes (mostly waiting)

[STEPS]
SECTION: PCR Pooling
4. Using a 10uL multichannel pipette, transfer 2 µL or  3 µL of PCR product from each row of your 96 well plate of PCR amplicons into the corresponding cells of a new eight tube strip. (Ex - If you are transferring 3 plates of amplications, at the conclusion, there should be 108 µL of product in each of the eight tubes in the strip [12 cells x 3uL x 3 plates].) Use 3 µLfrom each PCR reaction if you are combining three plates; 2 µL if you are combining 5 plates (see total volumes below in next step).

SECTION: PCR Pooling
5. Using a 200uL or 1000uL pipette, transfer the PCR pools from each of the eight tubes of the strip into a new 1.5mL LoBind eppi tube.

Final 1.5mL tube volumes:
3 plates - 288 samples - 864 µL (3uL per cell)
4 plates - 384 samples - 768 µL (2uL per cell) or 1.15 mL (3uL per cell)
5 plates - 480 samples - 960 µL (2uL per cell) 

Mix the tube by turning it upside down 3-5 times.
SECTION: Preparation
1. Bring magnetic beads to room temp. (Should be stored in the fridge)

SECTION: PCR Bead Cleanup
6. Subsample 500 µL of the amplicon pool to a new 1.5mL LoBind eppi tube. 

Retain the tube with the original product. You will run a gel electrophoresis comparison between this product and the purified product. 

SECTION: PCR Bead Cleanup
7. Vortex or shake beads thoroughly to suspend them in the solution. 
SECTION: PCR Bead Cleanup
8. Add 0.5X ratio of magnetic beads to the 1.5mL tube containing the pooled amplicons. Ex - for 250uL subsampled pool, add 125 µL of beads. For 500mL amplicon pool, add 250uL of beads.

Mix thoroughly by pipetting up and down 6-8 times.
SECTION: PCR Bead Cleanup
9. Incubate for 5 min at room temperature.

SECTION: PCR Bead Cleanup
10. Place sample tube on the magnetic separator for 3 min or until the solution clears. Beads should now be on the side of the tube.

SECTION: PCR Bead Cleanup
11. With the tube still on the magnet, remove the liquid from the tube and discard. Be sure not to disturb the beads.
SECTION: Preparation
3. Create a fresh batch of 80% ethanol. You will be using 2 mL in this protocol. You will be using more later, so make extra. A 15mL tube is one potential type of vessel.

8 mL 100% ethanol
2 mL molecular water

SECTION: PCR Bead Cleanup
12. With the tube still on the magnet, add 1000 µL of 80% ethanol to the tube and let sit for 2 min. Try to minimize disturbance of the beads. Fill gently with liquid stream from the pipette tip on opposite side of the beads.

SECTION: PCR Bead Cleanup
13. Remove ethanol by pipetting and discard.
SECTION: PCR Bead Cleanup
14.  Repeat the ethanol wash one time.    

SECTION: PCR Bead Cleanup
15. Dry by incubating the tube for 10-15 minutes at room temperature.  Ensure all of the ethanol has evaporated from the tube.
SECTION: Preparation
2. Heat a 1.5uL tube of molecular water to 55 °C in the heat block. ~1000uL should be sufficient in the tube. This step is optional but is helpful if a heat block is available.

SECTION: PCR Bead Cleanup
16. Remove the tube from the magnet and add 100 µL of 55 °C molecular water. Pipette up and down five times to mix until the pellet is fully suspended. 

The DNA will now be released from the beads and suspended in the water.

SECTION: PCR Bead Cleanup
17. Incubate for 2 min at room temperature.

SECTION: PCR Bead Cleanup
18. Place the tube back on the magnet for 3 min, or until the solution is clear.

SECTION: PCR Bead Cleanup
19. Transfer the water containing the DNA to a new 1.5mL LoBind eppi tube.

You should now have your pooled and purified DNA template.
SECTION: Gel Electrophoresis Validation
20. Run a gel with a lane of the original product and a lane of the purified DNA template side by side. 

Perform another purification if the primer band is still visible. 
SECTION: Quantification
21. If you have access to a Quantus/Qubit fluorometer, now is a good time to quantify the resulting amount of DNA in your purified sample. 

If not, the 100 µL of molecular water added above should put you near the right DNA concentration with room for a final adjustment.

You are looking to be around 1ug DNA per 50uL water as an end goal.


Promega Wizard Extraction - results in ~63 ng/uL
X-Amp extraction of dried tissues - results in ~72 ng/uL
X-Amp extraction of fresh tissues - results in ~73 ng/uL

Combination of all three resulted in ~69 ng/uL. There are 1000ng in a ug. 1000/69 = 14.5uL to get to 1ug. 


So for the final end product:

14.5uL of the resulting DNA solution combined with 35.5uL of water for the next step. (1ug DNA per 50uL water),