Difference between revisions of "Ligation and Transformation"

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'''Ligation and Transformation'''
+
'''Ligation'''
  
#Plan to do a ligation with the vector only and one with vector + insert
+
#Plan to do a ligation with the vector only and one with vector + insert.
#Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O
+
#Dilute vector to 20 ng/ul for use in the ligation, use Millipore pure dH2O.
#Calculate the amount of insert needed with the following formula
+
#Calculate the amount of insert needed with the following formula:
 
   10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
 
   10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
#Combine vector, insert, and pure dH2O in a total volume of 5 ul in a 1.5 ml tube
+
# Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
#Heat at 42 C for 2 minutes
+
# Add 1 ul 10X Ligation buffer and mix with pipetting.
#Add 5 ul 2X Quick Ligation buffer and mix with pipetting
+
# Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase]and mix with pipetting.
#Add 0.5 ul [http://www.neb.com/nebecomm/products/productM2200.asp Quick T4 DNA Ligase](and mix with pipetting
+
# Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
#Let stand 5 min at room temperature
+
#Let stand 5 min at room temperature.
#Thaw competent cells on ice (eg. 100 ul of  [http://www.zymoresearch.com/content/z-competent-e-coli-t3003-t3005-t3007-t3009-t3011-t3013-t3015-t3017 Z-competent JM109])
+
#Thaw competent cells on ice (eg. 100 ul of  [http://www.zymoresearch.com/content/z-competent-e-coli-t3003-t3005-t3007-t3009-t3011-t3013-t3015-t3017 Z-competent JM109]).
#Heat ligation at 65 C for 10 minutes
+
#Heat ligation at 65 C for 10 minutes.
#Place ligation tubes on ice for 2 minutes
+
#Place ligation tubes on ice for 2 minutes.
#Add at least 20 ul of competent cells to ligation using a cold pipette tip
+
 
#Let stand on ice for 5 minutes
+
'''Transformation'''
#Spread onto LB agar plates with the appropriate antibiotic
+
 
 +
#Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
 +
#Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
 +
#Let stand on ice for 5 minutes.
 +
#Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
 +
#Place your plates into the incubator.

Revision as of 19:33, 2 February 2017

Ligation

  1. Plan to do a ligation with the vector only and one with vector + insert.
  2. Dilute vector to 20 ng/ul for use in the ligation, use Millipore pure dH2O.
  3. Calculate the amount of insert needed with the following formula:
  10 ng vector x (insert size/vector size) x 3 = ng insert, eg. 10ng x (700 bp/2100 bp) x 3 = 10 ng
  1. Combine vector, insert, and pure dH2O in a total volume of 8 ul in a 1.5 ml tube
  2. Add 1 ul 10X Ligation buffer and mix with pipetting.
  3. Add 0.5 ul Quick T4 DNA Ligaseand mix with pipetting.
  4. Add 0.5 ul of the appropriate restriction enzyme. Your total volume in the tube should now be 10 uL.
  5. Let stand 5 min at room temperature.
  6. Thaw competent cells on ice (eg. 100 ul of Z-competent JM109).
  7. Heat ligation at 65 C for 10 minutes.
  8. Place ligation tubes on ice for 2 minutes.

Transformation

  1. Dilute competent cells using dilution buffer. Add 70 uL of buffer if you are doing two plates.
  2. Add half of your competent cell/buffer mix, 60 uL in the case of two plates, to your ligation.
  3. Let stand on ice for 5 minutes.
  4. Spread onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
  5. Place your plates into the incubator.