Ligation and Transformation - Revision history

Eckdahl at 21:39, 2 February 2017

2017-02-02T21:39:33Z

Eckdahl at 21:33, 2 February 2017

2017-02-02T21:33:08Z

Eckdahl at 18:50, 25 May 2012

2012-05-25T18:50:08Z

Eckdahl at 13:51, 16 June 2010

2010-06-16T13:51:26Z

Eckdahl at 17:03, 7 June 2010

2010-06-07T17:03:25Z

Eckdahl at 16:52, 7 June 2010

2010-06-07T16:52:39Z

New page

'''Ligation and Transformation'''

#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
#Calculate the amount of insert needed with the following formula
10 ng vector x (insert size/vector size) x 6 = ng insert
#
#
#

Go to the [http://partsregistry.org/Main_Page Registry ]and choose "Add a part"
# Decide whether the new part is a '''Basic Part''' (discrete function units that cannot be subdivided, like promoter, RBS, coding sequence), '''Composite Part''' (functional unit composed of basic parts, like gene expression cassette, inverter), or '''Construction Intermediate''' (no function, byproduct of construction).
#Choose the number for the next available part
#Be sure to include short and long descriptions of the part
#Once data is available on the part, go back and include it as "Experience"
<br>

@@ Line 16: / Line 16: @@
 '''Transformation'''
 #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.
+#Spread your mix onto LB agar plates with the appropriate antibiotic and mark the plates appropriately and legibly.
 #Place your plates into the incubator.

@@ Line 1: / Line 1: @@
-'''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:
 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

@@ Line 4: / Line 4: @@
 #Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O
 #Calculate the amount of insert needed with the following formula
-ng vector x (insert size/vector size) x 6 = ng insert, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng
+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
 #Heat at 42 C for 2 minutes

@@ Line 2: / Line 2: @@
 #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
+#Dilute vector to 10 ng/ul for use in the ligation, use Millipore pure dH2O
 #Calculate the amount of insert needed with the following formula
 ng vector x (insert size/vector size) x 6 = ng insert, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng
-#Combine vector, insert, and pure dH2O as a total volume of 5 ul in a 1.5 ml tube
+#Combine vector, insert, and pure dH2O in a total volume of 5 ul in a 1.5 ml tube
 #Heat at 42 C for 2 minutes
 #Add 5 ul 2X Quick Ligation buffer and mix with pipetting
@@ Line 12: / Line 12: @@
 #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
-#Place on ice for 1 minute
+#Place ligation tubes on ice for 2 minutes
-#Add competent cells to ligation (at least 20 ul)and wait 5 minutes
+#Add at least 20 ul of competent cells to ligation using a cold pipette tip
 #Spread onto LB agar plates with the appropriate antibiotic

@@ Line 4: / Line 4: @@
 #Dilute vector to 10 ng/ul for use in the ligation
 #Calculate the amount of insert needed with the following formula
-ng vector x (insert size/vector size) x 6 = ng insert
+ng vector x (insert size/vector size) x 6 = ng insert, eg. 10ng x (700 bp/2100 bp) x 6 = 20 ng
+#Combine vector, insert, and pure dH2O as a total volume of 5 ul in a 1.5 ml tube
+#Heat at 42 C for 2 minutes
+#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
-Go to the [http://partsregistry.org/Main_Page Registry ]and choose "Add a part"
+#Let stand 5 min at room temperature
-# Decide whether the new part is a '''Basic Part''' (discrete function units that cannot be subdivided, like promoter, RBS, coding sequence), '''Composite Part''' (functional unit composed of basic parts, like gene expression cassette, inverter), or '''Construction Intermediate''' (no function, byproduct of construction).
+#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])
-#Choose the number for the next available part
+#Heat ligation at 65 C for 10 minutes
-#Be sure to include short and long descriptions of the part
+#Place on ice for 1 minute
-#Once data is available on the part, go back and include it as "Experience"
+#Add competent cells to ligation (at least 20 ul)and wait 5 minutes
-<br>
+#Spread onto LB agar plates with the appropriate antibiotic