User manual

Droplet Digital
™
PCR Applications Guide | 43
Copy Number Variation Analysis
Lists of recommended restriction enzymes for CNV ddPCR are provided in Tables 4.2
and 4.3. Conditions for a typical restriction enzyme digestion are in Table 4.4. For more
information, visit the New England Biolabs, Inc. website (www.neb.com).
Table 4.2. Recommended restriction enzymes for CNV ddPCR (most-preferred 4-cutters).
Restriction
Enzyme Sequence
Digestion Buffer
(old)
Digestion Buffer
(new)
Incubation
Temperature, °C
CviQI G/TAC NEBuffer 2, 3, BSA 3.1 25
MseI T/TAA NEBuffer 2, 4, BSA CutSmart 37
AluI AG/CT NEBuffer 1, 2, 4 CutSmart 37
HaeIII GG/CC NEBuffer 2, 4 CutSmart 37
BsmI GAATGC(1/–1) NEBuffer 4 CutSmart 65
BstYI R/GATCY NEBuffer 2,4 2.1 60
BSA, bovine serum albumin.
Table 4.3. Recommended restriction enzymes for CNV ddPCR (most-preferred 6-cutters).
Table 4.4. Conditions for a typical restriction enzyme digestion.
Restriction
Enzyme Sequence
Methylation
Sensitivity
Digestion
(old)
Digestion
Buffer (new)
Incubation
Temperature, °C
HindIII A/AGCTT None NEBuffer 2 NEBuffer 2.1 37
EcoRI G/AATTC CpG NEBuffer
1, 2, 3, 4
EcoRI
buffer
37
Reaction Component Final Concentration Volume (50 µl/reaction), µl
10x digestion buffer 1x 5
gDNA 100 ng/µl Variable
Restriction endonuclease 10 U/µg DNA Variable
100x BSA (as needed) 1x 0.5
Water — Variable
BSA, bovine serum albumin; gDNA, genomic DNA.
Newly designed assays should be run across a thermal gradient (for example, 65–55°C) to
identify an annealing/extension temperature that optimizes separation between positive and
negative droplets while minimizing rain (droplets that fall between the major positive and
negative populations). If possible, select an annealing/extension temperature that optimizes
performance of both target and reference assays.
NEB, nuclear extraction buffer.