7.1 cDNA Amplification
-
Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.
cDNA Amplification Reaction Mix
Add reagents in the order listed
PN
1X (µl)
4X +
10% (µl)
8X +
10% (µl)
Amp Mix
2000047
50
220
440
cDNA Primers
2000089
15
66
132
Total
-
65
286
572
-
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.
-
Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly.
-
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
-
Store at 4°C for up to 72 h or −20°C for ≤1 week, or proceed to the next step.