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Richard Lab:Restriction Digest: Difference between revisions
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(New page: ==Overview== This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration: <center>'''-----EcoRI--XbaI--Part--SpeI--PstI----...) |
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==Procedure== | ==Procedure== | ||
# Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning | # Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning | ||
# Add the following in a micro-centrifuge tube | # Add the following in a micro-centrifuge tube | ||
**5μl of Buffer 2 | **5μl of Buffer 2 | ||
**1μl of BSA | **1μl of BSA | ||
**42μl of DNA solution (Dilute PCR products in half) | **42μl of DNA solution (Dilute PCR products in half) | ||
# Vortex Enzymes and add 20 units (1μl) of each to the tube | |||
# Vortex Enzymes and add 20 units (1μl) of each to the tube | |||
# Incubate reaction in a 37°C water bath for at least one hour (I like 1:45). | # Incubate reaction in a 37°C water bath for at least one hour (I like 1:45). | ||
**Longer digest gives more complete digestion, especially if you have >1µg of DNA, but can sometimes give nonspecific digestion | **Longer digest gives more complete digestion, especially if you have >1µg of DNA, but can sometimes give nonspecific digestion | ||
# Heat kill the digest for 15 minutes at 70°C. | # Heat kill the digest for 15 minutes at 70°C. | ||
# If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes | # If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes | ||
# Heat-kill enzymes at 70°C for 15 min. | |||
# Heat-kill enzymes at 70°C for 15 min. | |||
# Store digested DNA in the freezer (-20°C). | # Store digested DNA in the freezer (-20°C). | ||
