Annealing Oligos for Cloning - Revision history

Macampbell at 15:02, 4 July 2013

2013-07-04T15:02:25Z

2013-07-04T14:28:46Z

2012-06-27T14:13:06Z

Change in boiling time for annealing

2012-04-28T15:19:25Z

2012-04-27T19:53:28Z

2012-04-27T19:47:59Z

2012-01-21T14:51:44Z

2011-10-11T17:30:48Z

2011-10-11T17:30:16Z

2011-10-11T17:30:01Z

@@ Line 9: / Line 9: @@
   dH2O to 20 ul total
-# Boil 4 minutes in beaker with 400 ml H2O
+# Boil 4 minutes in beaker with 400 ml H<sub>2</sub>O
 # Turn off heat and let cool at least 2 hours

@@ Line 18: / Line 18: @@
   (__ ng vector)/(670 ng/nmol-base pair x __ base pairs x 10 exp-6 L) = ___ nM (nmol/L)
-  Example: (10 ng)/(670 x 2079 bp x 10 exp-6) = 7.2 nM
+  Example: (50 ng)/(670 x 2079 bp x 10 exp-6) = 36 nM
 Calculate the molarity of annealed oligos to be used for the ligation (assuming your will use 1 ul of the diluted oligos)
@@ Line 24: / Line 24: @@
   __ nM vector x (molar excess of oligos)
-  Example: 7.2 nM x 3 = 21.6 nM oligos
+  Ligation Example: 36 nM x 3 = 108 nM oligos
 Calculate how much to dilute annealed oligos
@@ Line 32: / Line 35: @@
   Dilution factor for oligos = 5000 nM / desired oligo molarity
-  Example: 5000 nM /21.6 nM = 231 dilution factor
+  Ligation Example: 5000 nM /108 nM = 46 dilution factor
 Dilute annealed oligos with dH20
-  Example: 1 ul annealed oligos plus 230 ul dH20
+  Ligation Example: 1 ul annealed oligos plus 45 ul dH20
 Use 1 ul of vector and 1 ul of diluted oligos for ligation

@@ Line 9: / Line 9: @@
   dH2O to 20 ul total
-# Boil 10 minutes in beaker with 400 ml H2O
+# Boil 4 minutes in beaker with 400 ml H2O
 # Turn off heat and let cool at least 2 hours

@@ Line 16: / Line 16: @@
 Calculate the molarity of the vector that will be used in the ligation (assuming you will use 1 ul of the vector)
-  (__ ng/ul vector)(10 exp6 ul/L)/(670 g/pmol x __ base pairs) = ___ nM
+  (__ ng vector)/(670 ng/nmol-base pair x __ base pairs x 10 exp-6 L) = ___ nM (nmol/L)
-  Example: (10 ng)(10 exp6/(670 x 2079 bp) = 7.2 nM
+  Example: (10 ng)/(670 x 2079 bp x 10 exp-6) = 7.2 nM
 Calculate the molarity of annealed oligos to be used for the ligation (assuming your will use 1 ul of the diluted oligos)

@@ Line 14: / Line 14: @@
 '''Dilute Oligos for Ligation'''
-Calculate the molarity of the vector that will be in the ligation
+Calculate the molarity of the vector that will be used in the ligation (assuming you will use 1 ul of the vector)
-  (__ ng vector)(10 exp6 ul/L)/(670 g/pmol x __ base pairs) = ___ nM
+  (__ ng/ul vector)(10 exp6 ul/L)/(670 g/pmol x __ base pairs) = ___ nM
   Example: (10 ng)(10 exp6/(670 x 2079 bp) = 7.2 nM
-Calculate the molarity of annealed oligos to be used for the ligation
+Calculate the molarity of annealed oligos to be used for the ligation (assuming your will use 1 ul of the diluted oligos)
   __ nM vector x (molar excess of oligos)

@@ Line 14: / Line 14: @@
 '''Dilute Oligos for Ligation'''
-Calculate how many pmol of vector will be in the ligation
+Calculate the molarity of the vector that will be in the ligation
-  (__ ng vector)/(.650 ng/pmol x __ base pairs)
+  (__ ng vector)(10 exp6)/(670 g/pmol x __ base pairs) = ___ nM
-  Example: (10 ng)/(.65 x 2079 bp) = .0074 pmol
+  Example: (10 ng)(10 exp6/(670 x 2079 bp) = 7.2 nM
-Calculate how many pmol of annealed oligos for the ligation
+Calculate the molarity of annealed oligos to be used for the ligation
-  __ pmol vector x (molar excess of oligos)
+  __ nM vector x (molar excess of oligos)
-  Example: .0074 x 6 = .0444 pmol/ul oligos
+  Example: 7.2 nM x 3 = 21.6 nM oligos
 Calculate how much to dilute annealed oligos
-  Annealed oligos are at 5 uM, or 5 pmol/ul
+ Stock oligos = 100 uM
-  Dilution factor for oligos = (5 pmol/ul)/(__ pmol/ul annealed oligos)
+  Annealed oligos = 5 uM = 5000 nM
-  Example: (5 pmol/ul)/(.0444 pmol/ul) = 113
+  Example: 5000 nM /21.6 nM = 231 dilution factor
 Dilute annealed oligos with dH20
-  Example: 1 ul annealed oligos plus 112 ul dH20
+  Example: 1 ul annealed oligos plus 230 ul dH20
-Use 1 ul of diluted oligos for ligation
+Use 1 ul of vector and 1 ul of diluted oligos for ligation