Richard Lab:Ligation

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (14:13, 29 March 2011) (view source)
(Procedure)
 
(7 intermediate revisions not shown.)
Line 1: Line 1:
 +
Back to [[Richard_Lab:protocols | Protocols]]
<center>
<center>
-
'''This protocol is for the typical ligation of DNA done using the [[Richard Lab:Bio-Brick Assembly Schedule|Richard Lab Standard Assembly 2.0]].   
+
'''This protocol is for the typical ligation of DNA done during [[Richard Lab:Amplified insert assembly|Amplified Insert Assembly]].   
'''The [[DNA_ligation | consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
'''The [[DNA_ligation | consensus Ligation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>
Line 8: Line 9:
::a. 11µl of deionized water
::a. 11µl of deionized water
::b.  2µl of Reaction Buffer for T4 Ligase.
::b.  2µl of Reaction Buffer for T4 Ligase.
-
::c.  3µl digested "Vector" (Phosphatased).  
+
::c.  2µl digested "Vector" (Phosphatased).  
-
::d.  3µl digested "Insert" (PCR product with DpnI).
+
::d.  4µl digested "Insert" (PCR product digested with DpnI).
::e.  1µl of [[T4_DNA_ligase|DNA Ligase]].
::e.  1µl of [[T4_DNA_ligase|DNA Ligase]].
2.  Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>
2.  Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)<br>
-
3.  Kill Reaction for 15 mins at 65°C
+
3.  Kill Reaction for 15 mins at 65°C '''Do not forget this step'''
===Notes===
===Notes===
-
* [[User:Michael A. Speer|Mike]] has had problems with blunt end ligation if this reaction is let go for too long.
 
* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.
* If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.
 +
 +
Back to [[Richard_Lab:protocols | Protocols]]

Current revision

Back to Protocols

This protocol is for the typical ligation of DNA done during Amplified Insert Assembly.

The consensus Ligation protocol should be consulted if deviating from the procedure outlined here.

Procedure

1.Mix together the following:

a. 11µl of deionized water
b. 2µl of Reaction Buffer for T4 Ligase.
c. 2µl digested "Vector" (Phosphatased).
d. 4µl digested "Insert" (PCR product digested with DpnI).
e. 1µl of DNA Ligase.

2. Incubate at room-temp for 10 mins (or at 4°C overnight for blunt ends)
3. Kill Reaction for 15 mins at 65°C Do not forget this step

Notes

  • If the ligase is not heat killed then it will remain attached to the DNA and will severely inhibit transformation efficiency.

Back to Protocols

Personal tools