General conditions for CAPS markers
Primers are used at 1 microM final concentration
dNTPs are used at 200 microM final concentration
In most cases 10 or 20 microliter reactions are suitable.
Genomic DNA should be used at 20-100 ng, depending on the DNA prep.
General PCR conditions:
35 cycles of:
94 degrees 30 seconds
52 degrees 30 seconds
72 degrees 1 minute
In some cases more cycles are required at annealing temperatures that differ from the standard temperatures. In some cases annealing temperatures may slightly vary depending on the PCR machine used
Generally digestions are performed with 10 microliters of PCR product.
As the reaction mix already contains some buffer, add 0.5 microliter of the appropriate buffer (no need to add no salt buffers (e.g. NEB buffer 1).
Add 0.5 microliter enzyme. In most cases this is sufficient, in rare cases less can be used.
Add water for the master mix: we add 4 microliters water per sample and pipet 5 microliter master mix to each well prior to adding the PCR product.
Digest for 1.5 to 2 hours at the indicated temperature.
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