Fusing
GFP to the Carboxy Terminus of Your Favorite Protein
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- Design the
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
Reaction
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
- 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.
- Pellet cells at 4°C at 5,000 x g for 5 minutes.
Decant supernatant.
- Wash cells with 5 ml of dH2O.
- Resuspend cells in 100 µl of 100 mM lithium
acetate (LiOAc).
- Transfer to two Eppendorf tubes.
- During cell preparation, boil sheared salmon
sperm carrier DNA for 5 min and then place on ice for at least 2 minutes.
- Pellet cells in microfuge for 15 sec. Decant
supernatant.
- Add IN THIS ORDER to
one tube:
- 240 µl of 50% PEG (mol. wt. 3350)
- 36 µl of 1.0 M LiOAc
- 25 µl of salmon sperm carrier DNA (10
mg/ml stock solution)
- 45 µl of cassette (from PCR reaction above)
- Mix. Place in 30°C rotator and leave for
45 minutes.
- Heat shock in 42°C water bath for 25 minutes.
- Pellet at 6,000 x g for 1 minute.
- Resuspend in 100 µl of dH2O
- Plate mix on 2 YPD plates and incubate at 30°C
overnight.
- Replica plate to the appropriate selective medium
(YPD G418 or SD -his) the following day.
- 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.
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