Silver: Restriction Digest

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(Digestion of BioBrick vector)
Line 15: Line 15:
#*700 ng BioBrick vector (BBa_V0002 or BBa_V0100)  
#*700 ng BioBrick vector (BBa_V0002 or BBa_V0100)  
#*1 µL 10 x BSA
#*1 µL 10 x BSA
-
#*1 µL 10 x 10x buffer (see [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website] for optimal double digest buffer choices)  
+
#*1 µL 10x buffer (see [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website] for optimal double digest buffer choices)  
#*0.2 µL enzyme 1 (20 units/µL)
#*0.2 µL enzyme 1 (20 units/µL)
#*0.2 µL enzyme 2 (20 units/µL)
#*0.2 µL enzyme 2 (20 units/µL)

Revision as of 14:28, 31 October 2005

Digestion of PCR product

  1. Mix:
    • All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
    • 3.5 µL 10x BSA
    • 3.5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
    • 0.2 µL enzyme 1 (20 units/µL)
    • 0.2 µL enzyme 2 (20 units/µL)
    • distilled water to 35 µL total volume
    • Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  2. Incubate at least an hour (better overnight) at 37 °C.
  3. Purify digested insert using PCR product purification kit.

Digestion of BioBrick vector

  1. Mix:
    • 700 ng BioBrick vector (BBa_V0002 or BBa_V0100)
    • 1 µL 10 x BSA
    • 1 µL 10x buffer (see NEB website for optimal double digest buffer choices)
    • 0.2 µL enzyme 1 (20 units/µL)
    • 0.2 µL enzyme 2 (20 units/µL)
    • distilled water to 10 µL total volume
    • Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.
  2. Incubate overnight at 37 °C.
  3. The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.
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