Team:Queens Canada/Notebook/Protocols/Ligation

From 2011.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 19: Line 19:
-
<body onLoad="runAccordion(6,225)">
+
<body onLoad="runAccordion(6,200)">
</div>
</div>
Line 77: Line 77:
<div id="rightcontenttext">
<div id="rightcontenttext">
-
<h3red> Ligation</h3red>
+
<h3red> (Quick) Ligation</h3red><p>
   
   
<regulartext> <b> Storage and Labelling </b> </regulartext> <br>
<regulartext> <b> Storage and Labelling </b> </regulartext> <br>
-
<regulartext> - Label the product tube as “EC” and in accordance with the standard labelling format, as outlined at the front of your lab book and on Google Docs.
+
<regulartext> - Store the ligation products in the -20⁰C freezer  </regulartext> <br>
 +
<regulartext> - Label the product tube as “DNA (AQ)” and in accordance with the standard labelling format, as outlined at the front of your lab book and on Google Docs </regulartext> <p>
-
</regulartext> <br>
 
-
<regulartext> - Label the product plate as “EC (resistance)” and in accordance with the standard labelling format, as outlined at the front of your lab book and on Google Docs. </regulartext> <p>
 
<regulartext> <b> Materials </b> </regulartext> <br>
<regulartext> <b> Materials </b> </regulartext> <br>
-
<regulartext> - Glass or plastic culture tubes </regulartext> <br>
+
<regulartext> - Linear vector DNA (20-100ng) </regulartext> <br>
-
<regulartext> - Growth medium containing appropriate antibiotics </regulartext> <br>
+
<regulartext> - Insert DNA (6:1 molar ration of insert:vector) </regulartext> <br>
-
<regulartext> - Glass pipette tubes </regulartext> <br>
+
<regulartext> - 5X T4 DNA Ligase (4µL) </regulartext> <br>
-
<regulartext> - Parafilm </regulartext> <p>
+
<regulartext> - Water, nuclease-free (15µL-vector and insert volume)</regulartext> <br>
 +
<regulartext> - Total volume (20µL)</regulartext> <p>
 +
 
<regulartext> <b> Procedure </b> </regulartext> <br>
<regulartext> <b> Procedure </b> </regulartext> <br>
-
<regulartext> 1. Flame a glass pipette (or use a sterile plastic pipette without flaming), open the bottle of medium and flame the mouth. <br>
+
<regulartext>1. Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase <br>
-
2. Withdraw amount you need to fill your tubes (5ml per tube), flame the cap and recap the bottle as quickly as possible. <br>
+
2. Transfer 5µl of the mastermix to each properly labeled sample tube. <br>
-
3. Remove the tube cap, flame the top of the culture tube, pipette in 5ml, flame the top of the tube and cap it. <br>
+
3. Add corresponding insert DNA and vector DNA into the tubes. Top the volume to 20µL using ddH2O. <br>
-
4. Pick up one colony using a P20 pipette tip. Uncap the tube, flame the top, inject the tip into the tube. <br>
+
4. Incubate one hour at 22⁰C (or room temperature) <br>
-
5. Gently vortex the tube to ensure that the bacteria in the tip mixes into the media. <br>
+
5. Heat inactivate T4 DNA ligase at 65⁰C for 10 min. <br>
-
6. Incubate the tubes at 37⁰C overnight or until cells have reached the desired concentration. This should take between 12 and 16 hours.<br>
+
6. Use up to 5 µL of the mixture for transformation of chemically competent cells.
-
7. When done, seal the transformed bacteria culture plate(s) that were used with Parafilm and store in 4⁰C fridge. <br>
+
 
-
<br>
+
  </regulartext> <br>
  </regulartext> <br>

Latest revision as of 20:33, 28 September 2011

Queen's
ligation
(Quick) Ligation

Storage and Labelling
- Store the ligation products in the -20⁰C freezer
- Label the product tube as “DNA (AQ)” and in accordance with the standard labelling format, as outlined at the front of your lab book and on Google Docs

Materials
- Linear vector DNA (20-100ng)
- Insert DNA (6:1 molar ration of insert:vector)
- 5X T4 DNA Ligase (4µL)
- Water, nuclease-free (15µL-vector and insert volume)
- Total volume (20µL)

Procedure
1. Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase
2. Transfer 5µl of the mastermix to each properly labeled sample tube.
3. Add corresponding insert DNA and vector DNA into the tubes. Top the volume to 20µL using ddH2O.
4. Incubate one hour at 22⁰C (or room temperature)
5. Heat inactivate T4 DNA ligase at 65⁰C for 10 min.
6. Use up to 5 µL of the mixture for transformation of chemically competent cells.

Retrieved from "http://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Ligation"