Team:WashU/Protocols/Ligation

From 2012.igem.org

(Difference between revisions)
(→General Ligation Protocol)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
<!--Thanks to NEB for the above instructions.  The original source can be found here: http://www.neb.com/nebecomm/products_intl/protocol658.asp-->
<!--Thanks to NEB for the above instructions.  The original source can be found here: http://www.neb.com/nebecomm/products_intl/protocol658.asp-->
-
{{WashUback}}
+
{{WashUbackprotocols}}
 +
<div id = "protocolshort">
=General Ligation Protocol=
=General Ligation Protocol=
-
1. Gather ingredients below and label PCR tube. (Total volume is 10ul)<br>
+
<dl>1. Gather ingredients below and label PCR tube. (Total volume is 10ul)<br>
-
      1ul 10x T4 ligase buffer<br>
+
<dd>1ul 10x T4 ligase buffer
-
      6:1 molar ratio of insert to vector (appx 10ng vector) <br>
+
<dd>6:1 molar ratio of insert to vector (appx 10ng vector)
-
      ddh20 (to bring total volume to 10ul)<br>
+
<dd>ddh20 (to bring total volume to 10ul)
-
      0.5ul T4 Ligase<br>
+
<dd>0.5ul T4 Ligase
 +
</dl>
2.  Add appropriate amount of deionized H2O to sterile 0.6 mL tube<br>
2.  Add appropriate amount of deionized H2O to sterile 0.6 mL tube<br>
3.  Add 1 μL ligation buffer to the tube. <br>
3.  Add 1 μL ligation buffer to the tube. <br>
Line 15: Line 17:
6. Add 0.5 μL ligase.  
6. Add 0.5 μL ligase.  
pipette up and down until well-mixed.  
pipette up and down until well-mixed.  
-
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting. DO NOT VORTEX T4 ligase is sensitive to shear)
+
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting. DO NOT VORTEX. (T4 ligase is sensitive to shear)
7. Let the 10 μL solution sit at 22.5°C for 30 mins<br>
7. Let the 10 μL solution sit at 22.5°C for 30 mins<br>
8. Denature the ligase at 65°C for 10min<br>
8. Denature the ligase at 65°C for 10min<br>

Latest revision as of 21:31, 9 August 2012


General Ligation Protocol

1. Gather ingredients below and label PCR tube. (Total volume is 10ul)
1ul 10x T4 ligase buffer
6:1 molar ratio of insert to vector (appx 10ng vector)
ddh20 (to bring total volume to 10ul)
0.5ul T4 Ligase
2. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
3. Add 1 μL ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. 4. Add appropriate amount of insert to the tube.
6. Add 0.5 μL ligase. pipette up and down until well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting. DO NOT VORTEX. (T4 ligase is sensitive to shear) 7. Let the 10 μL solution sit at 22.5°C for 30 mins
8. Denature the ligase at 65°C for 10min
9. Store at -20°C or use immediately
10. Transform 50ul of competent cells with 1-5ul reaction mixture

Modified from: [http://openwetware.org/wiki/Endy:DNA_ligation_using_T4_DNA_ligase Openwetware Endy Protocol]

Retrieved from "http://2012.igem.org/Team:WashU/Protocols/Ligation"