6.1 Sample Index PCR

Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run.

  1. Prepare Sample Index PCR Mix. Pipette mix and centrifuge briefly.

    2

     

    Fixed percentage added to total to help with pipetting

    Sample Index PCR Mix
    Add reagents in the order listed

    PN

    Volume for 1X (μl)

    Scaled Volume (μl)
    Amp Mix 2000047 50

     
    SI- PCR Primer B 2000128 7.5

     

    Total

    		 

    57.5

     

  2. Transfer 40 μl pre-amplified sample from step 5.3 to a new tube strip (35 μl of the remaining sample volume will be used for cDNA Amplification). Add 57.5 μl Sample Index PCR Mix to the sample. Pipette mix and centrifuge briefly.

  3. Add 2.5 μl of an individual Sample Index N, Set A to each well. Record assignment. Pipette mix and 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

    ~30 min

    Step

    Temperature

    Time

    1

    98°C

    00:00:45

    2

    98°C

    00:00:20

    3

    67°C

    00:00:30

    4

    72°C

    00:00:20

    Go to step 2, see table below for # cycles

    5

    72°C

    00:01:00

    6

    4°C

    Hold

    The table recommends a starting point for cycle number optimization based on Targeted Nuclei Recovery.

    Cycle Number Optimization Table

    Targeted Nuclei Recovery

    Total Cycles

    ≤2,000

    9

    2,001-6,000

    8

    >6,001

    7

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