Material

  • Set up the following reaction in a microcentrifuge tube on ice.(T4 DNA Ligase should be added last. )
  • Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)
  • Use NEBioCalculator to calculate molar ratios.

| Check | Reagent | Volume per 1 reaction | Memo |

ddH2O Up to 10
Backbone fragment X 0.020 pmol
Insert fragment Y 0.060 pmol
10x T4 ligase Buffer 1
T4 ligase 1 30 units. 12 units/µL
Total volume (µL) 10
  • The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
  • Gently mix the reaction by pipetting up and down and microfuge briefly.
  • For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
  • For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
  • Heat inactivate at 65°C for 10 minutes.
  • Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
Reaction Buffers
  • (T4 DNA Ligase Buffer (NEB #B0202) should be thawed on the bench or in the palm of your hand, and not at 37°C (to prevent breakdown of ATP).
  • Once thawed, T4 DNA Ligase Buffer should be placed on ice.
  • Ligations can be performed in any of the four standard restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase
  • Buffer (NEB #B0201) if they are supplemented with 1 mM ATP.
  • When supplementing with ATP, use ribo ATP (NEB #P0756). Deoxyribo ATP will not work.
  • Before ligation, completely inactivate restriction enzyme by heat inactivation, spin column (NEB #T1030), or Phenol/EtOH purification.



DNA
  • Heat inactivate (Antarctic Phosphatase, Quick CiP, rSAP) before ligation.
  • Keep total DNA concentration between 1-10 µg/ml.
  • Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Insert: vector molar ratio should be 6:1 to promote multiple inserts. Use NEBioCalculator to calculate molar ratios.
  • For cloning more than one insert, we recommend NEBuilder® HiFi DNA Assembly Products.
  • If you are unsure of your DNA concentration, perform multiple ligations with varying ratios.



Ligase
  • For most cohesive-end ligations, standard T4 DNA Ligase, Instant Sticky-End Ligase Master Mix, or the Quick Ligation Kit are recommended.
  • For blunt and single-base overhangs, Blunt/TA Ligase Master Mix is recommended.
  • For ligations that are compatible with electroporation, ElectroLigase (NEB #M0369) is recommended.
  • Standard T4 DNA Ligase can be heat inactivated at 65°C for 20 minutes.
  • Do not heat inactivate the Quick Ligation Kit or ligase master mixes.



Transformation
  • Add between 1-5 µl of ligation mixture to competent cells for transformation.
  • Extended ligation with PEG causes a drop off in transformation efficiency (Quick Ligation Kit).
  • Electroporation is recommended for large constructs (>10,000 bp). Dialyze sample or use a spin column (NEB #T1030) to purify first if you have used the Quick Ligation Kit or ligase master mixes.
  • For ligations that are compatible with electroporation, ElectroLigase is recommended.





Based on NEB protocol for T4 ligation. https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202

Dan Yamamoto-Evans 2019/11/24 14:25

Print at 96%.

  • interactome/t4ligation.txt
  • Last modified: 2019/11/24 15:04
  • by dan