7.1 cDNA Amplification

  1. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.

    2

     

    Fixed percentage added to total to help with pipetting

    cDNA Amplification Reaction Mix
    Add reagents in the order listed

    PN

    Volume for Master Mix (µl)

    Scaled Volume (µl)

    Amp Mix

    2000047

    50

     

    cDNA Primers

    		 2000089 15

     

    Total

    		 

    65

     

  2. Transfer 35 μl pre-amplified sample from step 5.3 to a new tube strip. Add 65 μl cDNA Amplification Reaction Mix to the sample.

  3. Pipette mix 15x (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

    ~30-45 min

    Step

    Temperature

    Time

    1

    98°C

    00:03:00

    2

    98°C

    00:00:15

    3

    63°C

    00:00:20

    4

    72°C

    00:01:00

    5

    Go to Step 2, see table below for total # of cycles

    6

    72°C

    00:01:00

    7

    4°C

    Hold

    The optimal number of cycles is a trade-off between generating sufficient final mass for library construction and minimizing PCR amplification artifacts. The number of cDNA cycles should also be reduced if large numbers of nuclei are sampled.

    Recommended starting point for cycle number optimization.

     

    Targeted Nuclei Recovery

     

    Total Cycles

    ≤2,000

    9

    2,001–6,000

    7

    ≥6,001

    6

  5. Store at 4°C for up to 72 h or −20°C for ≤1 week, or proceed to the next step.