Jamesh008:consensus DNA ligation protocol

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Contents

General Information

This is a consensus protocol click to see what this means.

DNA ligase is used to create a phosphodiester bond between the 5' phosphate and 3' hydroxyl groups of DNA. Most commonly, one needs to insert a DNA sequence of interest into a plasmid. Ideally, you can cut the DNA and vector with the same restriction enzyme. Then you can add both DNA sequences, the cut insert and vector sequence, and ligate them together to form a circular vector using DNA ligase. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques.

Materials

  • T4 DNA Ligase
  • 10x T4 DNA Ligase Buffer
  • Deionized, sterile H2O
  • Purified, linearized vector (likely in H2O or EB)
  • Purified, linearized insert (likely in H2O or EB)


Protocol Procedure

10μl Ligation Mix

Larger ligation mixes are also commonly used

  • 1.0 μL 10X T4 ligase buffer
  • 6:1 Molar ratio of insert to vector (~10ng vector)
  • Add (8.5 - vector and insert volume)μl ddH2O
  • 0.5 μL T4 Ligase

Calculating Insert Amount

 {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng}

This differs from the Knight calculation, not sure why, but it may be important.

Method

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. 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.
  3. Add appropriate amount of insert to the tube.
  4. Add appropriate amount of vector to the tube.
  5. Add 0.5 μL ligase.
    Vortex ligase before pipetting to ensure that it is 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 1 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Let the 10 μL solution sit at 22.5°C for 30 mins
  7. Denature the ligase at 65°C for 10min
  8. Dialyze for 20 minutes if electroporating
  9. Use disks shiny side up
  10. Store at -20°C

Notes

  1. Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)
  2. If you are having trouble with your ligation, NEB offers FAQ's (Quick Ligation T4 DNA ligase) to help.
  3. Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.
  4. Tom Knight has read that ligase can inhibit transformation. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation if you are having problems.
  5. Treating PCR products with proteinase K prior to restriction digest dramatically improves the efficiency of subsequent ligation reactions. [1]

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References

  1. Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, and Gewert D. . pmid:2011503. PubMed HubMed [Crowe-NAR-1991]

Specific Protocols

Endy:DNA ligation using T4 DNA ligase -- Using T4 DNA Ligase

Knight:DNA ligation using NEB Quick Ligation Kit -- 5min ligation.

Knight:TOPO TA cloning -- For PCR products.

Silver:Ligation -- A protocol using the Roche Kit.

BE.109:DNA ligation -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Contact

  • Jamesh008 is the Jamesh008:consensus DNA ligation protocol editor.
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