Team:Queens Canada/Notebook/Protocols/Ligation

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<classred1> <b> Rehydration </b></classred1>
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    <span class="classred"><a href="https://2011.igem.org/Team:Queens_Canada/Notebook/Protocols/Ligation">Ligation      </a></span>     </regulartext>
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<classred1> <b> ligation </b></classred1> </regulartext>
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<h3red> Rehydration- iGEM Standard Parts</h3red>
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<h3red> (Quick) Ligation</h3red><p>
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<regulartext> <b> Storage and Labelling </b> </regulartext> <br>
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<regulartext> - Store the ligation products in the -20⁰C freezer  </regulartext> <br>
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<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>
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<regulartext> <b> Materials </b> </regulartext> <br>
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<regulartext> - Linear vector DNA (20-100ng)  </regulartext> <br>
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<regulartext> - Insert DNA (6:1 molar ration of insert:vector) </regulartext> <br>
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<regulartext> - 5X T4 DNA Ligase (4µL) </regulartext> <br>
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<regulartext> - Water, nuclease-free (15µL-vector and insert volume)</regulartext> <br>
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<regulartext> - Total volume (20µL)</regulartext> <p>
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<regulartext> <b> Procedure </b> </regulartext> <br>
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<regulartext>1. Prepare a mastermix that contains T4 ligase buffer and T4 DNA ligase <br>
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2. Transfer 5µl of the mastermix to each properly labeled sample tube. <br>
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3. Add corresponding insert DNA and vector DNA into the tubes. Top the volume to 20µL using ddH2O. <br>
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4. Incubate one hour at 22⁰C (or room temperature) <br>
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5. Heat inactivate T4 DNA ligase at 65⁰C for 10 min. <br>
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6. Use up to 5 µL of the mixture for transformation of chemically competent cells.
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<h3red> Rehydration- Primers</h3red>
 
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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.

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