Richard Lab:Restriction Digest

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(Procedure)
(Procedure)
Line 6: Line 6:
# 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
+
# Heat kill the digest for 15 minutes at 75°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 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
+
-
# 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).

Revision as of 15:13, 12 June 2009

Contents

Overview

This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:

-----EcoRI--XbaI--Part--SpeI--PstI-----

Procedure

  1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning
  2. Add the following in a micro-centrifuge tube
    1. 5μl of Buffer 2
    2. 1μl of BSA
    3. 42μl of DNA solution (Dilute PCR products in half)
  3. Vortex Enzymes and add 20 units (1μl) of each to the tube
  4. Incubate reaction in a 37°C water bath for at least one hour (I like 1:45).
  5. Heat kill the digest for 15 minutes at 75°C.
  6. 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.
  7. Store digested DNA in the freezer (-20°C).

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. If you're not getting good digestion it might be because your enzyme is bad. Double the digestion time and see

References

If you are needing to know more see the other restriction digest protocols.

Contact

or instead, discuss this protocol.

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