Golden Gate Cloning with Oligonucleotide Inserts

Author: C. Goldbeck

Materials:

• 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

Critical steps:

• 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.

Procedure

Preparation of the Plasmid

1. 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.

2. Add 1 ul more of BsaI and digest for an additional 1 hr. at 37C.
3. Heat inactivate the BsaI and Antarctic phosphatase by heating the mixture to 65C for 20 min.
4. Do not purify the reaction, use it as is.

Prepare oligonucleotide inserts

5. 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

6. Heat inactivate the polynucleotide kinase by heating to 65C for 20 min.
7. Hybridize the oligonucleotides by adding 4uL 0.5 M NaCl per reaction.
8. 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.

Ligation & Transformation

9. Dilute dsDNA inserts 1/200 in water. They are already diluted 1/10 during the previous preparation.
10. 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.
11. 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
12. Gently thaw the chemically competent Mach 1 cells on ice.
13. For each reaction, aliquot 10ul of cells into pre-chilled microfuge tube.
14. Add 0.1 – 2 uL of ligation mixture
15. Incubate 30 min. on ice.
16. Heat shock at 42C for 30 sec.
17. Incubate on ice for 2 min.
18. Add 150 uL SOC media
19. Shake horizontally at 37C for 1 hr.
20. Pre-warm plates to 37C
21. Plate 100 uL / plate
22. Incubate overnight at 37C

See Plasmid Verification.