2/25/23, 1:27 PM Restricted Digest Protocol - Restricted Digest Design Tool | GenScript

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Restriction Digest Protocol

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Thaw all reagents on ice.

Assemble reaction mix into 50 µL volume in a microfuge tube.

Add reagents in following order: water, buffer, BSA, DNA template, restriction enzyme.

Gently mix by tapping tube. Briefly centrifuge to settle tube contents.

Prepare negative control reaction without template DNA.

Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice.

Typical Incubation time and temperature is 37°C for 1 hour, though time and temperature will vary depending on restriction
enzyme used.
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Restriction enzymes are typically inactivated by incubation at high temperature. Incubation time and temperature is 65°C for 20
min, though time and temperature will vary depending on restriction enzyme used.

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2/25/23, 1:27 PM Restricted Digest Protocol - Restricted Digest Design Tool | GenScript

Analyze the results of your PCR reaction via gel electrophoresis.

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Component Final Concentration/amount

water to 50 µL

buffer 1X

BSA 0.05 units/µL

DNA template 1 µg

Restriction enzyme 5-10 U per µg of DNA template (should not exceed 10% of reaction volume)

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2/25/23, 1:27 PM Restricted Digest Protocol - Restricted Digest Design Tool | GenScript

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