T4 cloning
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.
Tips
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
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