Fusing GFP to the Carboxy Terminus of Your Favorite Protein

PDF Version

Design oligonucleotide primers for polymerase chain reaction (PCR)

The same primers used for the pFA6a plasmid can be used for pDH3 and pDH5. See Wach et al., Yeast Vol. 13: 1065–1075 (1997) for a discussion of primer selection.
We typically use ~60 mer PAGE-purified oligos with ~40 bp homology to the gene of interest.

Forward primer: (5' to 3'):

(~40 bp upstream of stop codon)-GGTCGACGGATCCCCGGG

Reverse primer: (5' to 3' bottom strand):

(~40 bp downstream of stop codon)-ATCGATGAATTCGAGCTCG

Amplify the integration cassette from the plasmid

PCR:

A polymerase with relatively high fidelity and high activity is necessary. The Roche Expand Long Template PCR system has worked well for us. The following protocol assumes use of the Expand system.
We find it helps to linearize the plasmid with NotI before using it as a template for PCR. Only a 15 minute digestion is necessary.
For a 50 µl of PCR reaction, make two solutions:

Solution 1:

Reagent Volume (µl)
75 ng/µl linearized plasmid 1
100 pmole/µl forward primer 0.5
100 pmole/µl reverse primer 0.5
2.5 mM dNTP ( 2.5 mM each ) 10
dH2O 13

Solution 2:

Reagent Volume (µl)
Buffer #3 (provided) 5
Expand polymerase mix 0.75
dH2O 19.25
Mix solutions well individually. Combine and mix well again.
Run using the following PCR conditions:
  • 94°C for 2 minutes
  • 92°C for 10 seconds
  • 50°C for 30 seconds
  • 68°C for 4 minutes
Repeat previous three steps 9 times. Then,
  • 92°C for 10 seconds
  • 50°C for 30 seconds
  • 68°C for 4 minutes + 20 seconds per cycle
Repeat previous three steps 18 times. Then,
  • 68°C for 7 minutes
  • 4°C indefinitely
Run 5 µL on gel to confirm PCR reaction.

Transform yeast with the cassette

  1. Grow a 10 ml culture of diploid cells to ~90 Klett units (mid-log phase). This is enough for two transformations. Scale up as necessary.
  2. Pellet cells at 4°C at 5,000 x g for 5 minutes. Decant supernatant.
  3. Wash cells with 5 ml of dH2O.
  4. Resuspend cells in 100 µl of 100 mM lithium acetate (LiOAc).
  5. Transfer to two Eppendorf tubes.
  6. During cell preparation, boil sheared salmon sperm carrier DNA for 5 min and then place on ice for at least 2 minutes.
  7. Pellet cells in microfuge for 15 sec. Decant supernatant.
  8. Add IN THIS ORDER to one tube:
    1. 240 µl of 50% PEG (mol. wt. 3350)
    2. 36 µl of 1.0 M LiOAc
    3. 25 µl of salmon sperm carrier DNA (10 mg/ml stock solution)
    4. 45 µl of cassette (from PCR reaction above)
  9. Mix. Place in 30°C rotator and leave for 45 minutes.
  10. Heat shock in 42°C water bath for 25 minutes.
  11. Pellet at 6,000 x g for 1 minute.
  12. Resuspend in 100 µl of dH2O
  13. Plate mix on 2 YPD plates and incubate at 30°C overnight.
  14. Replica plate to the appropriate selective medium (YPD G418 or SD -his) the following day.
  15. After 2 days at 30°C, pick large colonies and streak for single colonies on selective medium.
NOTE: If transformation efficiency is low, try doubling the amount of carrier DNA or replacing your stock. In our experience, the carrier DNA is crucial for efficient transformation.
Finally one should confirm the correct integration by PCR.

Plasmids & Protocols