Tagging/Deleting Yeast Genes by PCR
1. Design PCR primers with 40 bp of sequence upstream/downstream of target site
- for C-terminal tagging, make sure that gene sequence is in-frame with the
tag
- for deletion, make sure that neighboring genes are not affected
- for GAL introduction/tagging, delete 50 bp of promoter and make sure
that gene sequence is in frame with tag
F1: 5’-40bp upstream of ATG-cggatccccgggttaattaa-3’ (coding seq.)
F2: 5’-40bp upstream of STOP-ggt cga cgg atc ccc ggg tt-3’ (coding seq.)
F4: 5’-90bp upstream of ATG-gaa ttc gag ctc gtt taa ac-3’ (coding seq.)
R1: 5’-40bp downstream of STOP-tcgatgaattcgagctcgt-3’ (reverse seq.)
R2: 5’-40bp downstream of ATG-cat ttt gag atc cgg gtt tt -3’ (reverse seq.)
R3: 5’-40bp downstream of ATG-gca ctg agc agc gta atc tg-3’ (reverse seq.)
R4: 5’-40bp downstream of ATG-acg cgg aac cag atc cga tt-3’ (reverse seq.)
R5: 5’-40bp downstream of ATG-ttt gta tag ttc atc cat gc-3’ (reverse seq.)
N-F: 5’-70bp upstream of ATG-cggccgccagg -3’ (coding seq.)
N-R: 5’-70bp downstream of ATG+ATG-ttt gta caa ttc atc cat acc atg -3’
(reverse seq.)
FLAG-F: 5’-40 bp upstream of STOP-agg gaa caa aag ctg gag-3’ (coding
seq.)
FLAG-R: 5’-40 bp downstream of STOP-ctatagggcgaattgggt-3’ (reverse seq.)
TAP-F: same as F2
TAP-R: same as R1
F5: 5’-40 upstream of STOP- ggtgacggtgctggttta (coding sequence)
R3: 5’-40bp downstream of STOP- -tcgatgaattcgagctcg (reverse sequence
2. Order oligos from IDT using the 100 nmol scale; have them purified by PAGE or
IE HPLC
3. Resuspend oligos in water at 25 M
4. Do PCR reactions
(do not use TRP1 template if using W303)
2 l diluted DNA template
2 l 25 M forward primer
2 l 25 M reverse primer
5 l 10X high fidelity polymerase buffer #2
0.5 l 25 mM dNTPs
0.5 l high fidelity polymerase
38 l H2O
94°C 5 min.
94°C 30 sec.
1 cycle
49°C 45 sec.
72°C 1.5-2.5 min.
35 cycles
72°C 7 min.
when using NATMX and HYGMX templates, add 2.5 l DMSO to
reactions and run the following program
94°C 2 min.
1 cycle
94°C 30 sec.
52°C 15 sec.
72°C 3 min.
35 cycles
72°C 20 min.
5. Check PCR by running 5 l out on 1% agarose gel
6. Transform entire PCR reaction into yeast using the LiOAc transformation
protocol
- be sure to transform log phase cells (you need high transformation
efficiency)
- use DMSO to increase transformation efficiency (48 microliters DMSO to
a standard LiOAc transformation)
- if the gene is essential, use a diploid!!! (and when in doubt, use a diploid)
- N-terminal tagging of essential genes must be done in a diploid since the
initial integration will disrupt gene expression
- remember a no DNA control
7. Plate transformations out onto YPD plates and incubate overnight at 30°C
8. Next day, replica plate to selective media and incubate 2-3 days at 30°C
9. Pick transformants to verify by PCR/western blotting
- I generally pick 10
10. For N-terminal tagging, integrate pURA3-GAL-cre/lox and select on –URA
plates
- once transformants are up, streak cells to –URA/gal-raff
- pick single colonies and score for loss of KAN resistance
- screen for protein by expression
Cassettes Available
No.
1
2
3
4
5
6
function
deletion
deletion
deletion
C-GFP
C-GFP
C-GFP
marker
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
PCR size
1559
1036
1403
2504
1981
2348
primers
F1/R1
F1/R1
F1/R1
F2/R1
F2/R1
F2/R1
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
C-3HA
C-3HA
C-3HA
C-13MYC
C-13MYC
C-13MYC
C-GST
C-GST
C-GST
deletion/GAL1
deletion/GAL1
deletion/GAL1
deletion/GAL1-3HA
deletion/GAL1-3HA
deletion/GAL1-3HA
deletion/GAL1-GST
deletion/GAL1-GST
deletion/GAL1-GST
deletion/GAL1-GFP
deletion/GAL1-GFP
deletion/GAL1-GFP
C-3FLAG
C-TAP
C-CFP
C-YFP
deletion
deletion
deletion
2C-TAP
4PA-TAP
pURA3-GAL-cre/lox
N-YFP
N-CFP
C-Cerelean
C-Venus
N-Venus
N-Cerelean
mCherry
2x-mCherry
mCherry
2x-mCherry
GFP (yeast codon)
2x-GFP (yeast codon)
3x-GFP (yeast codon)
Primers to check integration
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX6
TRP1
HIS3MX6
KANMX
HIS3MX
KANMX6
HIS3MX6
NATMX4
PATMX4
HYGMX4
KANMX
KANMX
URA3
KANMX
KANMX
KANMX6
HIS3MX6
KANMX
KANMX
HIS3MX
HIS3MX
URA3MX
URA3MX
URA3MX
URA3MX
URA3MX
1898
F2/R1
1375
F2/R1
1742
F2/R1
2325
F2/R1
1802
F2/R1
2169
F2/R1
2465
F2/R1
1942
F2/R1
2309
F2/R1
2061
F4/R2
1558
F4/R2
1925
F4/R2
2201
F4/R3
1678
F4/R3
2045
F4/R3
2763
F4/R4
2245
F4/R4
2612
F4/R4
2807
F4/R5
2284
F4/R5
2651
F4/R5
1500
FLAG-F/R
see notes on use
2504
F2/R1
2504
F2/R1
1300
F1/R1
1300
F1/R1
1600
F1/R1
F2/R1
F2/R1
N/A
N/A
2399
N-F/R
2399
N-F/R
2504
F2/R1
2504
F2/R1
2399
N-F/R
2399
N-F/R
F5/R3
F5/R3
F5/R3
F5/R3
F5/R3
F5/R3
F5/R3
KANMX6
HIS3MX6
NATMX4
HYGMX4
GGCCGGGTGACCCGGCGGGG
OSJ414
GCCTCCATGTCGCTGGCCGGG
CATGCCCCTGAGCTGCGCACG
OSJ415
OSJ416
Primers to switch markers
PR78
ccttgacagtcttgacgtgc
PR79
cgcacttaacttcgcatctg
OSJ417
OSJ418