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Richard Lab:Restriction Digest: Difference between revisions
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Richard Lab:Restriction Digest (view source)
Revision as of 21:23, 29 January 2010
, 29 January 2010→Procedure
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==Procedure== | ==Procedure== | ||
# Quickly vortex all ingredients (Buffer, BSA, DNA | # Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning. | ||
# Add the following in a micro-centrifuge tube | # Add the following in a micro-centrifuge tube: | ||
##5μl of Buffer | ##5μl of Buffer (usually NEBuffer 2) | ||
##1μl of BSA | ##1μl of BSA | ||
## | ##0.5 picomoles DNA | ||
# Vortex Enzymes and add 20 units | ##Water to make 48μl | ||
# Incubate reaction in a 37°C water bath for at least one hour | # Vortex Enzymes and add 1μl (20 units) of each to the tube. | ||
# Heat kill the digest for | # Incubate reaction in a 37°C water bath for at least one hour. | ||
# Heat kill the digest for 20 minutes at 80°C. | |||
# If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min. | # If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min. | ||
# Store digested DNA in the freezer (-20°C). | # Store digested DNA in the freezer (-20°C). | ||
