172
typically only added to the PCR when the denaturation
temperature has been reached to reduce mispriming):
5 μL 10× reaction buffer.
1 μL 10mM each dNTP solution.
1 μL primer SplB-ins forward (10uM stock).
1 μL primer SplB-ins reverse (10uM stock).
3 μL DMSO.
1–10 ng DNA template.
1 μL Pfu Turbo polymerase.
H 2 O added to a final volume of 50 μL.
3. The backbone is typically amplified using the following PCR
cycling protocol: 95 °C for 3 min, [95 °C for 10 s, 52 °C for
10 s, 68 °C for 7 min] × 30 cycles, 72 °C for 5 min.
4. Following PCR, purify the reaction using a commercial PCR
purification kit as per manufacturer’s instruction, and verify the
resulting DNA product by analytical agarose gel electrophoresis.
The DNA product should be ~7 kb when using Tem1-BLIP
inserted into the pET28a vector backbone.
5. Add a suitable amount of purified PCR product into an infusion
cloning reaction according to manufacturer’s instructions. The
complementary 5′ fifteen bases on both forward and reverse
primers will enable an inter-molecular infusion reaction that will
lead to the formation of the desired plasmid. Dilute 5× with
water and transform into One Shot TOP10 Chemically
Competent Cells as per manufacturer’s instructions before plating them on LB-Kan agar plates and incubating overnight.
6. Inoculate a few of the resulting colonies in LB-Kan liquid
medium overnight and prepare plasmid DNA using a commercial miniprep kit according to manufacturer’s instructions.
7. Sequence the resulting plasmids using primers pETF2 and
pETR and ensure the sequence is correct.
1. Transform the verified plasmid into SHuffle T7 Competent E.
coli as per manufacturer’s instructions. Inoculate the resulting
colonies overnight at 30 °C. The next day, dilute this culture at
1% into 800 mL LB-Kan liquid medium at 30 °C. Induce with
IPTG (0.4mM final) when OD reaches 0.5–0.8 followed by
overnight incubation at 16 °C. Pellet the cells by centrifugation. The cells can be frozen at −80 °C for later processing.
2. Resuspend the cells in 10 mL Lysis and Binding buffer in a
50 mL Falcon tube and place on ice. Lyse cells by sonication
for 10 min using the following pulsing sequence: 5 s sonication
at amplitude 35 followed by 10 s rest. Centrifuge at 4 °C at
10,000 × g for 10 min and collect the supernatant. Aspirate the
3.2 Protein
Expression
and Purification
Hui Chin Goh et al.
typically only added to the PCR when the denaturation
temperature has been reached to reduce mispriming):
5 μL 10× reaction buffer.
1 μL 10mM each dNTP solution.
1 μL primer SplB-ins forward (10uM stock).
1 μL primer SplB-ins reverse (10uM stock).
3 μL DMSO.
1–10 ng DNA template.
1 μL Pfu Turbo polymerase.
H 2 O added to a final volume of 50 μL.
3. The backbone is typically amplified using the following PCR
cycling protocol: 95 °C for 3 min, [95 °C for 10 s, 52 °C for
10 s, 68 °C for 7 min] × 30 cycles, 72 °C for 5 min.
4. Following PCR, purify the reaction using a commercial PCR
purification kit as per manufacturer’s instruction, and verify the
resulting DNA product by analytical agarose gel electrophoresis.
The DNA product should be ~7 kb when using Tem1-BLIP
inserted into the pET28a vector backbone.
5. Add a suitable amount of purified PCR product into an infusion
cloning reaction according to manufacturer’s instructions. The
complementary 5′ fifteen bases on both forward and reverse
primers will enable an inter-molecular infusion reaction that will
lead to the formation of the desired plasmid. Dilute 5× with
water and transform into One Shot TOP10 Chemically
Competent Cells as per manufacturer’s instructions before plating them on LB-Kan agar plates and incubating overnight.
6. Inoculate a few of the resulting colonies in LB-Kan liquid
medium overnight and prepare plasmid DNA using a commercial miniprep kit according to manufacturer’s instructions.
7. Sequence the resulting plasmids using primers pETF2 and
pETR and ensure the sequence is correct.
1. Transform the verified plasmid into SHuffle T7 Competent E.
coli as per manufacturer’s instructions. Inoculate the resulting
colonies overnight at 30 °C. The next day, dilute this culture at
1% into 800 mL LB-Kan liquid medium at 30 °C. Induce with
IPTG (0.4mM final) when OD reaches 0.5–0.8 followed by
overnight incubation at 16 °C. Pellet the cells by centrifugation. The cells can be frozen at −80 °C for later processing.
2. Resuspend the cells in 10 mL Lysis and Binding buffer in a
50 mL Falcon tube and place on ice. Lyse cells by sonication
for 10 min using the following pulsing sequence: 5 s sonication
at amplitude 35 followed by 10 s rest. Centrifuge at 4 °C at
10,000 × g for 10 min and collect the supernatant. Aspirate the
3.2 Protein
Expression
and Purification
Hui Chin Goh et al.