Plasmid construction
(1) PCR to get the target DNA fragment
A target DNA fragment was obtained by PCR, and the PCR condition was showed in (Table 1 and
Table 2 )
Table 1 The reaction system of PCR
Component
For 20 μL reation system
For 50 μL reation system
10×PCR buffer
2.0 μL
5.0 μL
dNTPs (2.5 mmol/L)
1.5 μL
4.0 μL
Primer-up (10 µmol/L)
1.0 μL
1.0 μL
Primer-down (10 µmol/L)
1.0 μL
1.0 μL
0.5 μL of plasmid or 1.0 μL of
1 μL of plasmid or 1.5 μL of
genome
genome
rTaq
0.5 μL
1.0 μL
ddH2O
Add to 20 μL
Add to 50 μL
Template
Table 2 The reaction program of PCR
Step
Procedure
Temperature
Time
Cycle
1
Initial denaturation
95 ℃
5 min
-
2
Denaturation
94 ℃
1 min
-
3
Primer annealing
Uncertainty a
1 min
-
4
Extension
72 ℃
Uncertainty b
-
5
Go to step 2, 3, 4
-
-
25-30 cycles
6
Eventually Extension
72 ℃
10 min
-
7
Storage ;
4℃
-
-
a: The primer annealing temperature is 5 C below Tm of primers and no lower than 40C
b: The extension time is about 1 min/kb of expected product
(2) plasmid extraction
plasmid was extracted using a plasmid extraction kit (Omega, Madison, United States).
(3) Enzyme digestion reaction of the plasmid and target DNA fragment
All of the restriction enzymes used come from TaKaRa, Dalian, China. The enzyme digestion reaction
was at 37 °C for 4 hours. The components of enzyme digestion system were showed in (Table 3).
Table 3 Components of enzyme digestion system
Components
Volume
10×Buffer
10 μL
Plasmid or target DNA fragment
40 μL
Restriction enzymes
5.0 μL + 5.0 μL
ddH2O
Add to 100 μL
(4) The connection of plasmid and target DNA fragment
The ligase used come from TaKaRa, Dalian, China. The connection reaction was in a 16 °C water bath
overnight. The connection system was showed in (Table 4).
Table 4 Connection system
Components
Volume
Solution I
5.0 μL
Target DNA fragment
4.0 μL or 3 μL
Plasmid
1.0 μL or 2 μL
(5) Make competent Escherichia coli strain DH5α cells
Competent E.coli DH5α cells were made using a competent cell creparation kit (TaKaRa, Dalian,
China).
(6) Transformation DNA fragments into competent E. coli
①Remove competent cells (E.coli DH5a) from -80 ℃ freezer; thaw on wet ice about 5 minutes.
②Add recombinant DNA fragment to the competent cells by moving the pipette through the cells
while dispensing. Gently tap tubes to mix.
③Incubate on ice for 30 min.
④Heat-shock cells 90 sec in a 42 ℃ water bath: do not shake.
⑤Place cells on ice for 2 min rapidly.
⑥Add 1 mL of LB medium to the tube containing cells, then incubate for 1 hours at 37 °C with
shaking speed around 100 rpm.
⑦Centrifuge at 13000 r/min for 1min and remove the supernatant.
⑧Pipette 500 µL of sterile H2O into the transformation tube to re-suspend the pellet.
⑨Plate 200 µL of the cell suspension onto the LB plate containing 100 mg/L Amp.
⑩Incubate the plates overnight at 37 °C.
Transformants were screened on LB plate containing 100 mg/L Amp. Plasmid was extracted. Enzyme
digestion reaction and electrophoresis were performed to identify the transformants.
2. Yeast transformation
Yeast transformation was carried out utilizing a lithium acetate procedure described previously [27].
The detailed procedure is describe as follow:
(1) Materials and Reagents
Deionized H2O
G418
PEG 3350
Lithium acetate dihydrate (LiAc)
Salmon sperm DNA
Transformation solution (see Recipes)
(2) Equipment
Water bath
Centrifuges
30°C shaker and incubator
Standard petri dishes
1.5 ml centrifuge tubes
(3) Recipes
Transformation solution: PEG 3350 [50% (w/v)] 240 µL
1 mol/L LiAc 36 µL
Salmon sperm DNA (2 g/L) 50 µL
Transforming DNA 34 µL
Total 360 µL
(4) Procedure
A: Make yeast competent cells
① Obtain yeast strains of interest and streak on YEPD plates. Let cells grow 2 d before inoculation.
②1st Inoculation: Inoculate one colony into 5 mL YEPD liquid medium. Grow cells overnight at
30 °C with shaking speed around 180 rpm.
③ 2nd inoculation. Transfer 500 µL of the cells culture into 4.5 mL YEPD medium. Grow cells at
30 °C for 4 h.
④ Transfer 1 mL of the cells culture medium above into a new 1.5 mL sterile eppendorf tube and
harvest cells by centrifugation at 10000 r/min for 1 min at 4 °C.
⑤Wash cells twice with 1 mL water.
⑥Wash cells with 1 mL of sterile 0.1mol/L LiAc buffer.
⑦ Re-suspend cells in 500 µL of sterile 0.1mol/L LiAc buffer.
⑧ Dispense 50 µL cells into a new 1.5 mL sterile eppendorf tube.
⑨ Centrifuge at 13000 r/min for 1 min at 4 °C to remove the supernatant.
B: Yeast cell transformation
①Make the transformation solution for the planned number of transformations plus one extra (negative
control) (see the recipes).
② Add the solution to the cell pellet, vortex to re-suspend the cells.
③ Incubate in a 30°C stilling incubator.
④ Heat shock for 40 min in a 40°C water bath.
⑤ Centrifuge at 13000 r/min for 1 min and remove the supernatant.
⑥ Pipette 1 mL of liquid YEPD into the transformation tube to re-suspend the pellet, then grow cells
for 4 hours at 30 °C with shaking speed around 100 rpm.
⑦Centrifuge at 13,000 r/min for 1min and remove the supernatant.
⑧Pipette 1 mL of sterile H2O into the transformation tube to re-suspend the pellet.
⑨Plate 200 µL of the cell suspension onto the YEPD plate containing 300 mg/L G418.
⑩ Incubate plates at 30 °C for 2~4 d.
Transformants were screened on YEPD plate containing 300 mg/L G418. PCR was applied to verify
the recombinant strains with accurate site integration.