Team:WashU/Protocols/Ligation

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=General Ligation Protocol=
=General Ligation Protocol=
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1.Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. (Upstream Part digestion: 2 µl),
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<dl>1. Gather ingredients below and label PCR tube. (Total volume is 10ul)<br>
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(Downstream Part digestion:2 µl), (Destination Plasmid digestion:2 µl), (10X T4 DNA Ligase Buffer:2 µl), (T4 DNA Ligase: 1 µl), (H2O:11 µl)
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<dd>1ul 10x T4 ligase buffer
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<dd>6:1 molar ratio of insert to vector (appx 10ng vector)
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<dd>ddh20 (to bring total volume to 10ul)
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<dd>0.5ul T4 Ligase
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</dl>
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2.  Add appropriate amount of deionized H2O to sterile 0.6 mL tube<br>
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3.  Add 1 μL ligation buffer to the tube. <br>
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Vortex buffer before pipetting to ensure that it is well-mixed.
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Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
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4. Add appropriate amount of insert to the tube.<br>
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6. Add 0.5 μL ligase.
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pipette up and down until well-mixed.
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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)
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7. Let the 10 μL solution sit at 22.5°C for 30 mins<br>
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8. Denature the ligase at 65°C for 10min<br>
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9. Store at -20°C or use immediately <br>
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10. Transform 50ul of competent cells with 1-5ul reaction mixture<br>
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2.Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.
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Modified from: [http://openwetware.org/wiki/Endy:DNA_ligation_using_T4_DNA_ligase Openwetware Endy Protocol]
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3.Transform 2 μl of the ligation product into 50 μl of competent E. coli cells (or other suitable host strain). Select using the antibiotic corresponding to the Destination Plasmid.
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[https://2012.igem.org/Team:WashU/Protocols Back to Protocols]

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]

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