User manual

42 | Droplet Digital
™
PCR Applications Guide
Considerations in planning a restriction digestion.
■
Do not cut the target or reference amplicon
■
Choose a methylation-insensitive enzyme
■
Read the chosen restriction enzyme FAQs on the manufacturer’s website. They often
describe known issues such as star activity
■
For most assays, a fragment size of 5 kb or less works fine. This can typically be
achieved with a 4-cutter or 6-cutter enzyme. For some assays, smaller template
fragment lengths (<500 bp) are required. This may be due to nearby inhibitory secondary
DNA structures/elements that get cut away with a smaller fragment size
■
RestrictionMapper (www.restrictionmapper.org) is a website that can help you
determine template fragment length and whether your designed amplicon is cleaved
by a given restriction enzyme
■
For human genomic DNA, use 10 U restriction enzyme/µg DNA
– Up to 20 U enzyme/µg DNA is acceptable. Higher concentrations of enzyme
could be required to resolve higher CN targets
■
Some ddPCR reactions are inhibited when high volumes of digestion buffer are included
because of high salt concentrations
– If possible, avoid use of NEBuffer 3 and 3.1, which have the highest
salt concentration
– Digested templates should be diluted a minimum of 10-fold in the final ddPCR
reaction setup to reduce the final salt concentration in ddPCR. For example,
no more than 2 µl of a 1x digest reaction should be loaded into a 20 µl ddPCR
reaction if possible
– Many assays will perform well regardless of which NEBuffer is used or the amount
of 1x digest reaction loaded into ddPCR
■
Incubate the reaction for 1 hr at the recommended temperature
■
Heat inactivation is not required, but can be considered if long-term storage of digested
template is required. Do not heat inactivate at greater than 65°C
■
DNA purification is not necessary after restriction digestion
■
Store digested DNA at –20°C
Copy Number Variation Analysis