Fields Lab
  • Research
    • DNA Technology
    • RNA Technology
    • Protein Technology
    • Phage Technology
    • Synthetic Biology
    • Previous Projects
    • Yeast Resource Center
  • Members
  • Publications
  • Resources
    • Cloning 6000 ORFs
    • PCR Primers
    • Recombination Cloning Protocols
    • Vector pOBD2
    • Vector pOAD
    • Strains Used in Two-Hybrid Screens
  • Contact

Recombination Cloning Protocols

Cloning by Co-Transformation and Recombination in Yeast

Required materials
• Linearized vector
• PCR product to be cloned
• Host yeast strain
• 50% PEG solution (MW=3350):
• 1M Lithium Acetate
• 1M Tris, pH 8.0
• 0.5M EDTA, pH 8.0
• boiled & cooled sheared salmon sperm DNA (10mg/ml)
• DMSO
• Yeast media and plates (depending on markers)

Procedure
1. Inoculate 50 ml YEPD with 0.3 ml liquid stock of PJ69-4a (or any other appropriate yeast strain; we use MATa for activation-domain fusions and MATalpha for baits) in a 250 ml Erlenmeyer flask and grow overnight at 30C (minimum 15 h, max. 24 h).

2. Spin out cells in 50 ml conical tube (3500 rpm, 3 min, RT), pour off supernatant, add 2 ml 0.1 M LiOAc and transfer resuspended yeast to 2 microfuge tubes. Spin out yeast (3500 rpm, 1 min, RT) and resuspend all this yeast (combine aliquots) in a total volume of 2ml 0.1M LiOAc.

3. Meanwhile make "1X PEG/LiOAc/TE" mix in a 50 ml tube:
                       16 ml 50% PEG
                       2ml 1M LiOAc
                       200ul Tris
                       40ul EDTA
                       500 ul boiled & cooled salmon sperm DNA
                       ~200 ng linear vector DNA
                        2.24 ml DMSO
                        Add enough water to bring up total volume to 20ml
                   ***Add the DMSO last and mix quickly after adding by shaking hard for 30 sec or so.

4. Add the 2ml yeast mix and mix hard by hand for 1 min or so. Immediately pour into trough and pipet 200 ul into each of 96 wells ( plus 2 controls) of a 96-well dish (e.g. Costar 3596).

5. Now add 3 ul rePCR products (PCR array shaken and spun before use) or 5 ul Yep351 (= positive control) or nothing ( = negative control). Seal with plastic or Aluminum tape and shake up and down THOROUGHLY.

6. Incubate 30 min at 30°C and heat shock at 42°C for 15 minutes. Let cool to room temp.
​
7. Spin 7 min at 2500 rpm and aspirate out the supernatant. Add 200 ul of water to all 96 wells and then resuspend and plate each well’s yeast one at a time onto 35 mm -Leu (or -Trp) plates. Leave growing at 30C for 1-3 days. Now record colony number and pick colonies.
Proudly powered by Weebly
  • Research
    • DNA Technology
    • RNA Technology
    • Protein Technology
    • Phage Technology
    • Synthetic Biology
    • Previous Projects
    • Yeast Resource Center
  • Members
  • Publications
  • Resources
    • Cloning 6000 ORFs
    • PCR Primers
    • Recombination Cloning Protocols
    • Vector pOBD2
    • Vector pOAD
    • Strains Used in Two-Hybrid Screens
  • Contact