6.1.1 Molecular cloning
Polymerase chain reaction (PCR)
The PCR was performed under the following condition using the specific primers and corresponding templates.
Component Concentration
Template 1 ng/µL
Primer fw 400 nM
Primer rev 400 nM
dNTPs 200 µM
HF buffer 1x
Phusion HF DNA polymerase 0.05 U/µL
The PCR program was as follows:
Step Temperature Time
Initial denaturation 98 °C 30 sec
Denaturation 98 °C 15 sec
Annealing 55 °C 30 sec
Elongation 72 °C 25 sec 30x
Final elongation 72 °C 5 min
Analytical and preparative agarose gel electrophoresis
0.8% or 2.5% agarose gel containing around 30 μg ethidium bromide (EB) was prepared. 4 µL PCR products were removed and mixed with 6x DNA loading dye. The DNA fragments were separated by the agarose gel electrophoresis in 1x TAE buffer at 110 V for 30 min. The gel was visualized under UV light by ChemiDoc XRS System.
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For the purpose of preparation, the DNA samples were resolved by 0.8% or 2.5% agarose gel electrophoresis. The desired bands were cut and treated with the Gel Extraction Kit. The concentration of recovered DNA was measured by Nanodrop and determined with the absorption at 260 nm.
Restriction enzyme digestion
The isolated PCR products (DNA insert) were double-digested by the restriction enzymes according to the manufacturer’s instruction. The reaction samples were gently mixed and incubated at 37 oC for 1 h. Meanwhile, the corresponding empty vectors were digested by the same restriction enzymes. As control, digest of the vectors by a single enzyme was performed under the same condition. Then, 10 U calf intestinal alkaline phosphatase (CIAP) was added to the digest reaction of the vectors, and the mixture was incubated at 37 oC for another 0.5 h.
The double-digested insert DNA and the vectors bearing the same sticky ends were ligated by T4 DNA ligase according to the manufacturer’s instruction. The samples were gently mixed and incubated at room temperature for 1 h. As control, the reaction without insert DNA was performed under the same condition.
Blunt-end cloning
The Blunt-end cloning was performed according to the manufacturer’s instruction. The synthesized blund-end DNA was inserted into the vector pJET1.2 via DNA ligation. The reaction sample on ice was mixed gently and then, incubated at 22 oC for 5 min.
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6.1.2 Competent cells and transformation
Chemically competent cells and transformation
NEB Turbo cells were inoculated to 50 mL SOB medium and incubated at 37 oC at 160 rpm overnight.
The pre-culture was transferred to 200 mL fresh medium of an initial OD600 value of 0.1. The culture was shaken for around 1.5 h to an OD600 of 0.5, and then seperated to 4 x 50 mL. Cells were harvested by centrifugation at 4 oC at 4400 rpm for 10 min, resuspended in 15 mL TfbI buffer of each tube, and incubated on ice for 20 min. Then cells were pelleted by centrifugation, resuspended and combined in 4 mL TfbII buffer, incubated on ice for 20 min. At last, cells were seperated into 100 μL and stored at -80 oC.
For the transformation, 100 μL cells were thawed on ice and mixed with 10 μL ligation reaction sample. After the incubation on ice for 0.5 h, cells were incubated at 43 oC for 45 sec, then on ice for an additional 2 min. Subsequently, cells were transferred to 1 mL pre-warmed SOC medium and the cultures were incubated at 37 oC at 800 rpm for 45 min. 200 μL cell cultures were spread on the LB-agar plate with carbenicillin and cultured at 37 oC overnight.
Electro-competent cells and transformation
Electro-competent cells were prepared from E. coli strains BL21 (DE3) and B834 (DE3). Cells were inoculated to 50 mL LB medium and incubated at 37 oC at 160 rpm overnight. The pre-culture was transferred to 500 mL fresh LB medium of an initial OD600 value of 0.1. The culture was shaken at 37
oC until the OD600 value reached 0.5. Cells were incubated on ice for 20 min and then, harvested at 4
oC at 4400 rpm for 10 min. The cell pellet was washed twice with 500 mL ice-cold sterile water and once with 50 mL ice-cold sterile 10% glycerol. At last, cells were resuspended in 5 mL of ice-cold sterile 10% glycerol, seperated into 100 μL and stored at -80 oC.
For the transformation, 100 μL cells were thawed on ice and mixed with 1 μL plasmid. After the incubation on ice for 5 min, cells were transferred into a 1 mm pre-cooled GenePulser electroporation cuvette, and treated by GenePulser Xcell with the E. coli program (1 mm, 1.8 kV).
Subsequently, cells were transferred to 1 mL pre-warmed SOC medium and the cultures were incubated at 37 oC at 800 rpm for 45 min. 200 μL cell cultures were spread on the LB-agar plate with carbenicillin and cultured at 37 oC overnight.
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Single colonies on the carbenicillin-containing LB-agar plate were inoculated individually to 5 mL LB medium with carbenicillin and incubated at 37 oC at 160 rpm overnight. 10 μL culture was diluted with 100 μL sterile water. PCR in a final volume of 20 μL was performed under the following condition. The PCR products were analyzed by 2.5% agarose gel electrophoresis.
Component Concentration
pJET1.2 FP 200 nM
pJET1.2 RP 200 nM
Diluted culture 9.2 μL
Taq DNA polymerase Master Mix 1x
The PCR program was as follows:
Single colonies on the carbenicillin containing LB-agar plate were inoculated individually to 5 mL LB medium with carbenicillin and incubated at 37 oC at 180 rpm overnight. Cells were harvested by centrifugation at 4 oC at 4400 rpm for 10 min. The plasmid was extracted by Miniprep Kit and eluted by water. The concentration of the isolated plasmid was measured by Nanodrop.
Sequencing was performed by GATC Biotech using around 500 ng plasmid mixed with 2.5 µM primer.
Sequence data was analyzed by Clonemanage8.
Glycerol stock for cell storage
The sequenced plasmid was transformed to the desired electro-competent E. coli strain BL21 (DE3) or B834 (DE3). Cells containing the desired plasmid were cultivated on the LB-agar plate with carbenicillin.
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A single colony was inoculated to 5 mL LB medium with carbenicillin and incubated at 37 oC at 180 rpm overnight. The culture was seperated to 500 µL and mixed with 500 µL glycerol in a 1.5 mL tube.
The glycerol stock was frozen in the liquid nitrogen and stored at -80 oC.