Golden Gate Cloning with Oligonucleotide Inserts
Author: C. Goldbeck
- BsaI, NEB catalog # R0535, note: Do not use BsaIHF
- Antarctic Phosphatase, NEB catalog #M0201
- Polynucleotide kinase, NEB #
- T4 ligase, NEB #
- T4 ligase buffer, NEB #
- Mach 1 chemically competent cells
- HF BsaI
- HF BamHI
- Use Antarctic phosphatase, not calf alkaline phosphatase (CIP). CIP cannot be heat inactivated, while Antarctic phosphatase can be.
- Do not purify the digested plasmid.
- Use the regular T4 DNA ligation buffer. The fast digest versions contain PEG, which appears to interfere with the reaction.
Preparation of the Plasmid
- Digest and dephosphorylate the plasmid, by mixing:
- 14 uL plasmid
- 3 uL 10x NEB buffer #4
- 3 uL 10x Antarctic buffer
- 0.2 uL 100xBSA
- 2 uL BsaI, 2U/uL
- 1 uL Antarctic phosphatase, 2U/uL
- 6.8 uL water
Be sure to use the standard BsaI, not the HF enzyme. Digest with 1 ul of enzyme for 1 hr. at 37oC.
- Add 1 ul more of BsaI and digest for an additional 1 hr. at 37C.
- Heat inactivate the BsaI and Antarctic phosphatase by heating the mixture to 65C for 20 min.
- Do not purify the reaction, use it as is.
- Phosphorylate the oligonucleotides by mixing as below and then incubating the resulting mixture for ~1.5 hrs at 37C.
- 3 uL 100 uM sense oligonucleotide
- 3 uL 100 uM antisense oligonucleotide
- 3 uL 10x T4 DNA ligase buffer
- 2 uL polynucleotide kinase (PNK)
- 18 uL water
- Heat inactivate the polynucleotide kinase by heating to 65C for 20 min.
- Hybridize the oligonucleotides by adding 4uL 0.5 M NaCl per reaction.
- Then place in boiling water bath for 2 min., remove water bath from heat source (still in the water bath) and allow to cool to room temperature.
- Dilute dsDNA inserts 1/200 in water. They are already diluted 1/10 during the previous preparation.
- Prepare 10 uL of a solution containing a 1:3 molar ratio of dephosphorylated, cut plasmid DNA to oligonucleotide insert with a total of 100 ng of DNA.
- Set up ligation reaction by mixing as below and incubate at room temperature for 20 min. Use the standard NEB T4 DNA ligase kit, not the rapid kit.
- plasmid DNA + dsDNA insert to 10 uL
- 2 uL 10x T4 DNA ligase buffer
- 1 uL T4 DNA ligase
- Gently thaw the chemically competent Mach 1 cells on ice.
- For each reaction, aliquot 10ul of cells into pre-chilled microfuge tube.
- Add 0.1 – 2 uL of ligation mixture
- Incubate 30 min. on ice.
- Heat shock at 42C for 30 sec.
- Incubate on ice for 2 min.
- Add 150 uL SOC media
- Shake horizontally at 37C for 1 hr.
- Pre-warm plates to 37C
- Plate 100 uL / plate
- Incubate overnight at 37C
Prepare oligonucleotide inserts
Ligation & Transformation
See Plasmid Verification.