~Use this protocol after samples have pooled using Pooling Protocol~
Clean and prepare workspace:
Sterilize pipettes with a dilute bleach and then an ethanol wipe-down
Bleach and wipe down the lab bench.
print out plate map
Use the Pooling Protocol to pool samples based on relative band strength.
Vortex and spin pooled plates. Put on ice.
Gather primer strip tubes, Qiagen PCR master mix (MM), molecular-grade water from freezer. Defrost, vortex well, spin down, and nest into an ice bucket. If you are preparing the index primers for the first time, you should be diluting them to 1:20 from the stock primers.
Note: It is important to take special care with the index primers. Only expose them to a limited number freeze-thaw cycles, and don’t leave them at RT for extended periods.
Using the printed plate map, mark or tape off wells of a semi-skirted PCR plate that will not receive template DNA. [This is only relevant if you had gDNA template wells that were empty and therefore have TS-PCR wells that are empty].
label PCR plate appropriately
Make a mix of 5uL PCR master mix and 2.5uL H2O per sample (plus 1-2 samples worth of cushion) in a 1.5mL tube. Vortex and spin.
Dispense 7.5uL of this solution into each untapped well of the semi-skirted PCR plate.
Arrange primer strip tubes in a plate holder like this for convenience. For more guidance on setting up these plates, see this detailed tutorial here.
F1
R1
…
R12
F2
…
F8
Transfer 1uL of each F (forward) and R (reverse) primers to the plate using the multichannel pipette.
Now your index plate has the MM-water solution and the unique index combinations for each well of 96-well plate
Make sure to note the index combination (forward and reverse primers) for each well. If you do not know the index combination for each well you will not be able to demultiplex your samples and distinguish between them after sequencing.
Use the multichannel pipette to transfer 0.7uL of PCR product from the first round of PCR (TS-PCR product) across all wells.
Heat seal with foil lid.
Set up thermocycler program to match the table below. Launch thermocycler. Collect samples in ~1hr.
Lid 105C, vol 10uL
Step
Temperature
Time
1
95C
15:00
2
94C
0:30
3
55C
1:30
4
72C
1:30
5
Return to step2, 6X
6
72C
10:00
7
12C
∞
Before sequencing:
Use Gel Electrophoresis Protocol to check that the fragments are now ~170bp longer (XXXbp total for MCO and XXXbp total for ARF1)
Pool all of the product of the second round index PCR together into a 1.5 ul tube. You should have one 1.5 ul tube for each of your projects. A project should be defined ahead of time based on the amplicon fragment size and number of samples.
Transfer 50 ul of the library pool to another tube. Dilute with PCR grade water by one half (add an equal volume of water). Clean with a 1x ratio of SPRI beads (i.e., 50 ul of beads). Submit or do fragment analysis (note you need more than one pool ~11 to make a chip at the EGL worth the money). Submit or do qPCR with Illumina standards.
