8.5 Sample Index PCR

  1. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x sample index name (PN-3000431 Dual Index Plate TT Set A well ID) used.

  2. Add 50 μl Library Amp Mix to 30 µl sample.

  3. Add 20 μl of an individual Dual Index TT Set A to each well and record the well ID used. Pipette mix 5x (pipette set to 90 μl). Centrifuge briefly.

  4. Incubate in a thermal cycler with the following protocol.

    Lid Temperature

    Reaction Volume

    Run Time

    hh:mm:ss

    105°C

    100 µl

    ~25-40 min

    Step

    Temperature

    Time

    1

    98°C

    00:00:45

    2

    98°C

    00:00:20

    3

    54°C

    00:00:30

    4

    72°C

    00:00:20

    5  

      Go to step 2, see below for # of cycles

    6

    72°C

    00:01:00

    7

    4°C

    Hold

    The total cycles should be optimized based on 25% carry forward cDNA yield/input calculated during cDNA QC & Quantification

    Recommended cycle numbers

    cDNA Input

    Total Cycles

    0.25-50 ng

        

    14-16

    50-250 ng

    12-14

    250-600 ng

    10-12

    600-1,100 ng

    8-10

    1,100-1,500 ng

    6-8

    >1500 ng

    5

  5. Store at 4°C for up to 72 h or proceed to the next step.