Silver: Quick & Dirty Digest & Ligation

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(Quick Digestions)
Current revision (14:55, 3 July 2007) (view source)
(Quick Digestions)
 
Line 12: Line 12:
#*2 uL 10x appropriate NEB buffer
#*2 uL 10x appropriate NEB buffer
# Insert: Mix (to a total of 10 uL):  
# Insert: Mix (to a total of 10 uL):  
-
#*7.2 uL mini-prepped 'insert' DNA
+
#*7 uL mini-prepped 'vector' DNA (I use 7 ul if using V0120 eluted into 30ul MP)
 +
#*8.2 uL distilled water
#*0.4 uL restriction enzyme 1 at 20 units/uL
#*0.4 uL restriction enzyme 1 at 20 units/uL
#*0.4 uL restriction enzyme 2 at 20 units/uL
#*0.4 uL restriction enzyme 2 at 20 units/uL
-
#*1 uL 10x BSA
+
#*2 uL 10x BSA
-
#*1 uL 10x appropriate NEB buffer
+
#*2 uL 10x appropriate NEB buffer
#Incubate overnight at 37C.
#Incubate overnight at 37C.
# Gel purify the insert using a Qiagen spin column. Elute using 35 uL.
# Gel purify the insert using a Qiagen spin column. Elute using 35 uL.

Current revision

Quick & Dirty Digest & Ligation

  • Note: This procedure is NOT recommended for beginners. Beginners should start with the restriction digest and ligation procedures. While in the recommended procedures the DNA concentration is measured and adjusted to optimal levels, in this procedure it is not measured and estimated concentrations are used guide the amounts used. Beginners frequently have significant variations in DNA concentration between mini-preps, which can substantially lower the ligation efficiency. (I.e. You may not get any correct transformants & you'll have to do it again anyway.)

Quick Digestions

  1. Vector: Mix (to a total of 20 uL):
    • 7.5 uL mini-prepped 'vector' DNA (I use 7 ul if using V0120 eluted into 30ul MP)
    • 7.5 uL distilled water
    • 0.3 uL restriction enzyme 1 at 20 units/uL
    • 0.3 uL restriction enzyme 2 at 20 units/uL
    • 0.4 uL calf intestinal alkaline phosphatase (CIP)
    • 2 uL 10x BSA
    • 2 uL 10x appropriate NEB buffer
  2. Insert: Mix (to a total of 10 uL):
    • 7 uL mini-prepped 'vector' DNA (I use 7 ul if using V0120 eluted into 30ul MP)
    • 8.2 uL distilled water
    • 0.4 uL restriction enzyme 1 at 20 units/uL
    • 0.4 uL restriction enzyme 2 at 20 units/uL
    • 2 uL 10x BSA
    • 2 uL 10x appropriate NEB buffer
  3. Incubate overnight at 37C.
  4. Gel purify the insert using a Qiagen spin column. Elute using 35 uL.
  5. PCR purify the vector. Elute in 35 uL.

Quick Ligation

  1. Mix:
    • 1 uL digested vector
    • 3 uL digested insert
    • 4 uL distilled water
  2. Using the Roche Quick Ligation Kit, subsequentially add (vortex to mix in between additions):
    • 2 uL reagent #2
    • 10 uL reagent #1
    • 1 uL reagent #3
  3. Ligate for 15 minutes at room temperature.
  4. Transform 2 uL of ligation mix in 12.5 uL TOP10 competent cells.

Last edited by Caroline Ajo-Franklin 03/27/2007.

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