TITLE: ITS1 Amplicon Prep
AUTHORS: aglazer

[DESCRIPTION]
This protocol was provided by IDT in the the xGen ITS1 Amplicon Panel manual.

[STEPS]
SECTION: Prepare panel-specific Multiplex PCR Reaction Mix
1. Before mixing reagents, calculate the total volume of the Master Mix based on the number of reactions required, with 15% overage for pipetting. 
SECTION: Prepare panel-specific Multiplex PCR Reaction Mix
2. Make the Multiplex PCR Reaction Mix according to the table below and pipette-mix the Master Mix thoroughly.

 


 


 



SECTION: Prepare panel-specific Multiplex PCR Reaction Mix
3. Use a multichannel to pipette out 20 µL the PCR Mastermix into each well of a mid-skirt plate as needed and keep ON ICE until template addition.

SECTION: Perform Multiplex PCR
4. Pre-program the thermal cycler for multiplex PCR according to the table below and allow the block to reach 98oC before loading samples


 


 


SECTION: Perform Multiplex PCR
5. Use a multichannel to add 10 µL of template to the 20 µL Multiplex PCR Reaction Mix and pipette-mix thoroughly.

SECTION: Perform Multiplex PCR
6. Place the mid-skirt plate containing template and Master Mix in the thermal cycler and run the Multiplex PCR Program.


 

SECTION: Perform Post-Multiplex PCR Cleanup
10. Make sure that beads and samples are at room temperature. Spin down the sample plate and briefly vortex beads to homogenize before use.
SECTION: Perform Post-Multiplex PCR Cleanup
11. Add 30 µL of magnetic beads (ratio: 1.0x) to each 30 uL sample after thermal cycling and mix by vortexing. Pulse-spin the samples in a microcentrifuge tube to collect contents.

 


SECTION: Perform Post-Multiplex PCR Cleanup
12. Incubate the samples for 5 min minutes at room temperature, off the magnet. After incubation, place the sample tubes on a magnetic rack until the solution clears and a pellet is formed (about 5 min minutes).
SECTION: Perform Post-Multiplex PCR Cleanup
13. While the sample is on the magnet, remove and discard the supernatant without disturbing the pellet (about 5 µL may be left behind) using a clean pipette tip. Leave the plate on the magnet.

SECTION: Perform Post-Multiplex PCR Cleanup
14. Add 180 µL of freshly prepared 80% ethanol solution to the pellet while it is still on the magnet. Use care not to disturb the pellet. Incubate for 30 s seconds, then carefully remove the ethanol solution. 

SECTION: Perform Post-Multiplex PCR Cleanup
15. Repeat the previous step, for a second wash with the ethanol solution. 
SECTION: Perform Post-Multiplex PCR Cleanup
16. Pulse-spin the samples in a microcentrifuge, place back onto the magnet, and remove any residual ethanol solution from the bottom of the tube with a small volume tip.
SECTION: Perform Post-Multiplex PCR Cleanup
17. Resuspend each bead pellet in 19.4 µL post-PCR TE Buffer off-magnet and pipette mix. 
SECTION: Prepare Indexing PCR Master Mix (while the samples are on the thermal cycler)
7. Before mixing reagents, calculate the total volume of master mix based on the number of reactions required, with 15% overage for pipetting. It is recommended to prepare at least 10 reactions at any one time to maintain a volume of Enzyme I3 that can be accurately pipetted.
SECTION: Prepare Indexing PCR Master Mix (while the samples are on the thermal cycler)
8. Prepare the Indexing Master Mix ON ICE according to the following table

 

 

SECTION: Prepare Indexing PCR Master Mix (while the samples are on the thermal cycler)
9. Set aside the Indexing MMX on ice until template addition.
SECTION: Perform Indexing PCR
21. Add 3.7 µL of the pre-mixed xGen Amplicon UDI primer pair to each sample (17.4 µL).

SECTION: Perform Indexing PCR
22. Add 28.9 µL of the cold indexing PCR reaction mix, stored on ice, to each sample. Mix thoroughly by pipetting (total volume will be 50 µL).

SECTION: Perform Indexing PCR
20. Program the thermal cycler with the Indexing PCR program described in the table below and allow the block to reach 37oC before loading samples.

 


 

 

SECTION: Perform Indexing PCR
23. Spin down the sample plate, place in the thermal cycler, and run the Indexing PCR program.
SECTION: Perform Post-Indexing PCR Cleanup
24. Make sure the beads are at room temperature. Spin down the sample plate and briefly vortex the beads to homogenize before use.
SECTION: Perform Post-Indexing PCR Cleanup
25. Add 42.5 µL of fresh magnetic beads (ratio: 0.85x) to each 50 uL sample after thermal cycling and mix by vortexing.

 

SECTION: Perform Post-Indexing PCR Cleanup
26. Incubate the samples for 5 min minutes at room temperature, off the magnet. Pulse-spin the samples in a microcentrifuge. Afterwards, place the sample tubes on a magnetic rack until the solution clears and a pellet is formed (about 5 min minutes).
SECTION: Perform Post-Indexing PCR Cleanup
27. While the sample is on the magnet, remove and discard the supernatant without disturbing the pellet (about 5 µL may be left behind) using a clean pipette tip. Leave the plate on the magnet.
SECTION: Perform Post-Indexing PCR Cleanup
28. Add 180 µL of freshly prepared 80% ethanol solution to the pellet while it is still on the magnet. Use care not to disturb the pellet. Incubate for 30 s seconds, then carefully remove the ethanol solution. 

SECTION: Perform Post-Indexing PCR Cleanup
29. Repeat the previous step, for a second wash with the ethanol solution. 
SECTION: Perform Post-Indexing PCR Cleanup
30. Pulse-spin the samples in a microcentrifuge, place back onto the magnet, and remove any residual ethanol solution from the bottom of the tube with a small volume tip.
SECTION: Perform Post-Indexing PCR Cleanup
31. Immediately add 22 µL of post-PCR TE buffer and resuspend the pellet by pipetting up and down until homogenous. Incubate for 2 min minutes off the magnet.
SECTION: Perform Post-Indexing PCR Cleanup
32. Place the samples back on the magnet and transfer the clean 20 µL library eluates to a fresh plate. Make sure that the eluate does not contain magnetic beads (indicated by brown color in eluate). If magnetic beads are present, place the plate on the magnet, wait for pellets to re-form, then transfer eluate again.

SECTION: Perform Post-Indexing PCR Cleanup
33. SAFE STOP: Store freshly prepared libraries at -20oC.
SECTION: Perform Post-Multiplex PCR Cleanup
18. Incubate the samples for 5 min minutes at room temperature, off the magnet. After incubation, place the sample tubes on a magnetic rack until the solution clears and a pellet is formed (about 5 min minutes).
SECTION: Perform Post-Multiplex PCR Cleanup
19. While the sample is on the magnet, remove 17.4 µL of the supernatant without disturbing the pellet using clean pipette tips and transfer to a new plate.