Toronto/Lab Protocols/Ligation

From 2007.igem.org

Latest revision as of 03:37, 27 October 2007

Ligation

1. Calculate the concentration of DNA in both the plasmid and insert from the previous step. The conversion factor for the HindIII ladder we use is 0.10 (because the HindIII concentration is 100ng/μL), so use that to convert the value read off the HindIII ladder chart.
2. Calculate how much plasmid and insert you need. To ligate the two parts, the insert is added at 3 times the molar concentration of the plasmid. Use 10-15 ng of plasmid. Remember: the plasmid to insert ratio must be 1:3. Aim for a total volume of 10μL (this includes ligase, buffer, and ddH₂O).
   • Quantity of insert = (quantity of plasmid x 3)/(plasmid size/insert size)
3. Your recipe should now look like this:
   • Plasmid (calculated in step 2)
   • Insert (calculated in step 2)
   • 1 μL of 10x buffer
   • 0.5 μL of ligase
   • top with ddH₂O (to make 10 μL total volume)
4. In a 1.5 mL Eppendorf tube, add plasmid, insert, and ddH₂O.
5. Heat tube to 45 °C for 5 minutes to melt any cohesive termini.
6. Sit tube on ice for 5 minutes.
7. Add ligase buffer and ligase to the Eppendorf tube.
8. Gently vortex and spin down the tubes for 10 seconds to ensure all of the reagents are at the bottom of the tube.
9. Incubate the solution overnight at 16 °C or for 4 hours at 20 °C (Depending on how much time you have that day). It may be possible to incubate at room temperature for two hours if pinched for time.
10. Heat inactivate the ligase at 45 °C for 10 minutes.
11. Transform newly formed part into cells and plate them with the proper antibiotics (Check Spreadsheet).
12. Start the cloning process all over again!

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