Agarose Gel Electrophoresis
Material
TAE buffer
Agarose Powder
Ethidium Bromide (EB)
Comb and Bed
Step (take 30ml gel for instance)
① Weigh 0.3g agarose powder and 30μl 1%TAE buffer, then add to a flask;
② Heat up until the solution is completely dissolved. If it boils, move away from the heat until it cools down and put it back on the heat until it absolutely dissolved.
③ While heating, prepare the bed where the gel polymerizes. Make sure that it is well balanced and tight, and that the comb is well placed.
④ When the solution cools down to approximately 55 ℃ , add 1µl EB to the homogeneous
solution and mix well.
⑤ Pour the solution into the bed and clear all the bubbles in it with a tip.
⑥ When the gel solidification solidifies, carefully pull out the “comb”;
⑦ Place the gel in the electrophoresis chamber, then add enough TAE buffer till there is about 2-3 mm of buffer over the gel.
⑧ Mix the samples with loading buffer in a 10:1 ratio, then put the samples into the wells, as
well as marker into the first well.
⑨ Run the gel at 120V for about 30 minutes, then check the result under the Blue Light Gel
Imager.
Note
① For different size of gels, we have 25 ml,50 ml and 100 ml agarose gel.
② We often use 0.7%,1% and 1.5% agarose gel for different samples.
Double Enzyme DNA Digestion& Agarose Gel Electrophoresis
Material
Plasmid DNA
10× FastDigest or 10× FastDigest Green Buffer
Double Distillation Water (ddH2O)
FastDigest enzyme
Alkaline phosphatase
Steps
① Add 1μg plasmid DNA, 1μl FastDigest enzyme, 2μl 10× FastDigest or 10× FastDigest
Green Buffer and ddH2O to 20μl into an PCR tube.
② Put the tube into the oven at 37℃ for 1h to 1.5h.
③ Add 1μl Alkaline phosphatase.
④ Put the tube into the oven at 37℃ for 0.5h.
⑤ Stop the reaction by placing the tube at -4°C for 5 to 10 minutes.
⑥ Agarose Gel Electrophoresis.
⑦ Store the tube into the refrigerator at -20℃.
Gel Purification
Material
TIANGEN Universal DNA Purification Kit
Gel with DNA sample
Double Distillation Water (ddH2O)
Centrifuge
Shaker
Water Bath
Steps
① Cut the gel that DNA sample lies with a knife.
② Put the gel into an EP tube, add PC solution, which has the same volume as the gel.
Shake the tube at 50℃ for 10min till the gel is completely dissolved and the solution
color is yellow.
③ Insert the adsorption column into a collection tube, add 500μl BL equilibrium solution,
centrifuge at 12,000G for 1min, discard the filtrate and reuse the collection tube.
④ Add the solution to the adsorption column, centrifuge at 12,000G for 1min, discard the
filtrate and reuse the collection tube.
⑤ Add 600μl PW rinse solution, let stand for 5min, centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.
⑥ Repeat step⑤.
⑦ Put the adsorption column into a clean EP tube, centrifuge at 12,000G for 2min, discard
the filtrate. Open the cover of adsorption column and dry it out in the air for 5min.
⑧ Add 100μl ddH2O (preheated to 70 ℃ in advance), let stand for 2min, and centrifuge
at 12,000G for 2 min.
⑨ Add filtrate to the adsorption column again, let stand for 2min, and repeat step⑧.
⑩ Store the filtrate -20 ℃.
Note
Weight the gel before adding PC solution, 0.1g gel approximately equal to 100μl solution.
Infusion
Material
5 × CE MultiS Buffer
Exnase MultiS
Double Distillation Water (ddH2O)
Primer
Steps
① Calculate the amount of DNA required for the recombination reaction according to the formula.
② Prepare the following reaction system on ice:
a. X/Y calculates the amount of vector and the amount of each insert according to the formula.
b. Negative control-1 can be used to confirm whether there is a circular plasmid residue in the
linearized cloning vector, and it is recommended.
c. Negative control-2 is recommended when the insert amplification template is a circular
plasmid with the same resistance as the cloning vector.
It is recommended that the circular plasmid residue detection of the linearized vector and
insert be performed independently.
d. Positive control reactions can be used to exclude the influence of other experimental
materials and operating factors.
③Use a pipette to gently mix (do not shake to mix), and centrifuge briefly to collect the reaction
solution to the bottom of the tube.
④React at 37°C for 30 min; drop to 4°C or cool on ice immediately.
Note
① In order to ensure the accuracy of sample addition, the linearized vector and insert can be
properly diluted before preparing the recombinant system, and the sample amount of each
component is not less than 1 μl.
②Calculation of vector and fragment usage:
Optimum usage per segment=(0.02×Fragment base pairs) ng (0.03 pmol)
For example, when the inserts with lengths of 0.5 kb, 1 kb, and 2 kb are cloned into a cloning
vector with a length of 5 kb, the optimal amount of the vector and each fragment to be used is:
Optimum use amount of linearized cloning vector: 0.02 × 5000 = 100 ng;
Optimum usage of 0.5 kb insert: 0.02 × 500 = 10 ng;
Optimum usage of 1 kb insert: 0.02 × 1000 = 20 ng;
Optimum use of 2 kb insert: 0.02 × 2000 = 40 ng
a. The amount of linearized cloning vector should be between 50 - 200 ng. When using the
above formula to calculate the optimal amount of DNA to be used beyond this range, simply
select the minimum/maximum amount of use.
b. The amount of each insert should be greater than 10 ng. When using the above formula to
calculate the optimum usage amount is lower than this value, just use 10 ng directly.
c. When the linearized cloning vector and the amplified product of the insert fragment are used
directly without DNA purification, the total volume added should not exceed 1/5 of the
volume of the reaction system, that is, 4 μl.
Overlap PCR & Agarose Gel Electrophoresis
Material
2 x Phanta Max Master Mix
Primer
Double Distillation Water (ddH2O)
Loading Buffer
DNA Marker
Steps
① Adding 25μl of 2 x Phanta Max Master Mix(5μM), 1μl per fragment, 4μl of primer
mix, then add ddH2O up to 50μl into a PCR tube. Different group use specific tube
with distinctive sign.
② Place those PCR tubes into Peltier thermal cycler.
③ Set the protocol as follow: begin at 95℃ for 30 secs, then keep 95℃ for 15 secs
for denaturation, decrease to 60℃ for 15 secs, 72℃ for 1 min/kb and repeat that
cycle 30 times, finally maintain 16℃ infinity.
④ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.
Check the result under the Blue Light Gel Imager.
Note
As we use Green Taq Mix, we don’t need to add Loading when Agarose Gel Electrophoresis.
PCR & Agarose Gel Electrophoresis – Colony PCR
Material
Green Taq Mix, Primer, Double Distillation Water (ddH2O), Eight tube centrifuge, EP Tube,
Tweezers, plastic wrap, Shaker, PCR Eight-Tube, DNA Marker
Steps
(Take 5 tubes of eight tube centrifuge for example)
1) Pick bacteria
① Add 1mL Amp LB to 1.5mL EP Tube.
② Use a small tip with tweezers to pick out a single clone and put them into the EP
Tube.
③ Put the EP tube on the Rank and wrap it with plastic wrap.
④ Insert it into the shaker obliquely, shake for 2h.
2) Colony PCR（after shaking for 2h）(take eight tubes for instance)
① Adding 6.6μl Primer Mix(5μl), 41.3μl 2 x Green Taq, 29.1μl of ddH2O to the first
tube of eight-tube, then draw 9ul from the first tube to the next 4 tubes.
② Centrifuge the eight-tube in a centrifuge for several seconds.
③ Take the eight-tube into Clean bench.
④ Add 1μl Bacteria Liquid.
⑤ Set the protocol as follow: begin at 95℃ for 3 mins, then keep 95℃ for 15 secs
for denaturation, decrease to 55℃ for 15 secs, 72℃ for 1 min and repeat that
cycle 30 times, 72℃ for 1 min, finally maintain 4℃ infinity.
⑥ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.
Check the result under the Blue Light Gel Imager.
⑦ Select the right System for Shaking bacteria and Plasmid Extraction.
Note
① The lid of the PCR Eight-tube is placed in the ultra-clean workbench to make sure
we add Bacteria Liquid at the ultra-clean workbench.
② As we use Green Taq Mix, we don’t need to add Loading when Agarose Gel
Electrophoresis.
PCR & Agarose Gel Electrophoresis
Material
2 x Phanta Max Master
Mix Primer
Double Distillation Water (ddH2O)
Loading Buffer DNA
Marker
Steps
① Adding 25μl of 2 x Phanta Max Master Mix, ណng Template DNA, 4μl of primer
mix, then add ddH2O up to 50μl into a PCR tube. Different group use specific tube
with distinctive sign.
② Place those PCR tubes into Peltier thermal cycler.
③ Set the protocol as follow: begin at 95℃ for 30 secs, then keep 95℃ for 15 secs
for denaturation, decrease to 60℃ for 15 secs, 72℃ for 1 min/kb and repeat that
cycle 30 times, finally maintain 16℃ infinity.
④ Adding all the samples to the hole. Run the gel at 120V for about 25 minutes.
Check the result under the Blue Light Gel Imager.
Plasmid conservation
Material
Cryopreservation tube
50% Glycerin
Alcohol lamp
Steps
① Add 400μl 50% Glycerin and 600μl bacterial liquid
② Mix it upside down and put it in the refrigerator at -80 degrees
Note
Burn the mouth and cap of the flask with an alcohol lamp before and after opening the
glycerin bottle.
Plasmid Extraction
Material
TIANprep Mini Plasmid Kit II
Absolute Ethanol
Double Distillation Water (ddH2O)
Centrifuge
Steps
① Insert the adsorption column into a collection tube, add 500μl BL equilibrium solution,
centrifuge at 12,000G for 1min, discard the filtrate and reuse the collection tube.
② Add the bacterium liquid to a centrifuge tube, centrifuge at 12,000G for 1min, remove
the supernatant as much as possible, keep the precipitate in the tube.
③ Add 500μl solution P1 (the RNase A need to be added in advance) into the centrifuge
tube. Use a pipette or vortex oscillator to completely suspend the bacterial cell
precipitate.
④ Add 500μl solution P2 into the centrifuge tube and turn it up and down gently for 6-8
times till the cells are fully lysed, when the liquid should be clear and sticky, meanwhile
12,000G for 10min.
⑥ Add the supernatant collected in the previous step into the adsorption column (the
adsorption column is put into the collection tube). Centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.
⑦ Add 500μl solution PD to the adsorption column. Centrifuge at 12,000G for 1min,
discard the filtrate and reuse the collection tube.
⑧ Add 600μl solution PW (the absolute ethanol need to be added in advance) to the
adsorption column. Centrifuge at 12,000G for 1min, discard the filtrate and reuse the
collection tube.
⑨ Repeat step⑧.
⑩ Centrifuge the adsorption with collection tube at 12,000G for 2min, discard the filtrate
and reuse the collection tube. Open the cover of adsorption column and dry it out in the
air for 5min.
⑪ Place the adsorption column into a clean centrifuge tube. Add 200μl ddH2O to the
middle part of the adsorption membrane, placed it at room temperature for 2-5 min,
and centrifuge at 12,000G for 2min. The plasmid solution was collected into the
centrifuge tube.
Note
① Add RNase A to solution P1 before using.
② After adding solution P2, do not shake the tube violently to avoid pollution.
③ In step⑥, pay attention not to draw the white precipitate in the centrifuge tube out.
④ Add absolute ethanol to solution PW before using.
⑤ In step⑪, in order to increase the recovery efficiency of plasmid, the solution obtained
in the centrifuge tube can be readded into the centrifugal adsorption column and repeat
step⑪ again.
Protocol for LB medium preparation
Material
LB Broth Agar Powder, Miller LB Broth Powder, Miller Ultrapure (UP) Water, Double
Distillation
Water (ddH2O), Ampicillin ·
Steps
(1) Making LB liquid medium
① Weigh 12.5g of LB Broth Powder.
② Add the powder into 500ml UP Water.
③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.
(2) Making LB solid medium
① Weigh 12g of LB Broth Agar Powder.
② Add the powder into 300ml UP Water.
③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.
④ Pour the medium into culture dishes in the ultraclean worktable.
(3) Making LB selective medium
① Weigh 12g of LB Broth Agar Powder.
② Add the powder into 300ml UP Water.
③ Autoclave entire bottle of LB medium under 121 Celsius degree for 20 minutes.
④ Weigh 0.98g of Ampicillin.
⑤ Add the ampicillin into 9.8ml ddH2O. The ddH2O is sterilized previously. Shake the
mixture fully.
⑥ Filter the mixture through a filter to remove microorganism.
⑦ Add 1μl of mixture into an EP tube with 999μl ddH2O. The ddH2O is sterilized previously.
⑧ When the LB medium cool down to nearly 50℃, add 300μl antibiotic mixture into it in
the ultraclean worktable, then pour it into culture dishes
Transformation
Material
Competent Cell
Spreader
Plasmid (with target gene)
Ice Maker
Water Bath
Shaker
Oven
LB Liquid Medium
LB Selective Medium
Steps
① Take 100μl competent cell suspension from -80 ℃ refrigerator into an EP tube and put
it in ice bath until it thaws.
② Add 1μl plasmid with target gene into the suspension and put it in ice bath for 30min.
③ Put the EP tube into 42 ℃ water bath for 90sec.
④ Transfer the tube into ice bath for 3min immediately.
⑤ Add 900μl LB liquid medium into the tube, then shake it with 300rpm at 37 ℃ for 1h.
⑥ 5krpm, 5min Centrifuge
⑦ Discard the 850μl supernatant and resuspend the remaining 150μl
⑧ Apply 150μl bacterium liquid in a LB selective medium plate, then flip it and put it in
oven at 37℃ for 16h.
Note
① The whole process is operated under sterile condition.
② Switch on the ice maker and the water bath before beginning.
③ The time of step ③ and ④ must be controlled precisely.
Protocol for Cell Transfection and Stimulation
Material
96-well cell culture plates (or 24-well cell culture plates)
DMEM
Complete medium (High Glucose DMEM Medium supplemented with 10% FBS and 1% Penicillin Streptomycin Solution)
Polyethyleneimine (PEI) solution (1 mg/mL)
Steps
①Seed approximately $1×10^4$ cells into 96-well cell culture plates ($5×10^4$ for 24-well cell culture plates).
②Culture for 16h before transfection.
③Total plasmid mixes of 100ng per well (500ng for 24-well cell culture plates) are mixed thoroughly in serum-free DMEM before PEI solution is added into the plasmid mixture in a ratio of 1:3 (plasmid weight/PEI weight).
④The plasmid - PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for 6-8 hours.
⑤Cells are changed into fresh medium and applied with stimulus before sampling and analysis assay.
Note
To validate and optimize the gene circuits to reach the intended functionality and performance with high throughput, transient transfections are preferred. Also, easily detected reporters (such as SEAP and Luciferase) are also preferred during this process to evaluate the dynamics of the circuits.
Protocol for Cell Culture and Subculture
Material
PBS
0.25% Trypsin EDTA solution
Complete medium (High Glucose DMEM Medium supplemented with 10% FBS and 1% Penicillin Streptomycin Solution)
Steps
(1)Cell culture
①Culture in a humidified incubator at 37°C, 5%CO2.
②Check the color medium; healthy usually leaves medium slightly orange; observe the cells under a phase microscope.
③If the cells are not very confluent, remove the growth medium and replace it with a fresh complete medium.
④If the cells are at least 70%-80% confluence, subculture them.
(2)Subculturing cells (60 mm × 15 mm cell culture plate)
①Aspirate the growth medium from the cells and wash the cells with 2ml PBS to clear the FBS.
②Aspirate the PBS, then add 1ml 0.25% trypsin EDTA solution.
③Incubation for several minutes at 37°C and check the culture with an inverted microscope to ensure the cells are rounded up, indicating they are detached from the surface.
④Add 1ml fresh complete medium, then pipet all the adherent cells into cell suspension and pipet up and down.
⑤Transfer the cell suspension into a 15ml centrifuge tube and centrifuge at 720g for 5 minutes.
⑥Aspirate the filtrate, then add 2ml fresh medium and resuspend the cell.
⑦Place 9.5 ml of fresh medium in each new culture. Add 500ul cell suspension to each of the fresh plates that have been appropriately labeled.
⑧Culture in a humidified incubator at 37°C, 5%CO2.
Note
The time of treating cells with 0.25% trypsin EDTA solution should not be too long. Otherwise, it will cause toxicity to cells and affect cell status
Protocol for SEAP assay in vitro
Material
Chemiluminescence-based SEAP assay kit (e.g., Sigma Aldrich; cat. no. 1177984200)
buffer (2x): 20 mM L-Homoarginine hydrochloride, 1 mM MgCl2, 21% (v/v)
diethanolamine, pH 9.8.
Substrate for SEAP: 120 mM para-nitrophenyl phosphate (pNPP) in 2SEAP buffer.
Transparent 96-well plates.
Colorimetric plate-reader.
Steps
① Sample 200 μL culture medium from each well, heat inactivate at 65 C for 30 min.
② During the heat inactivation procedure, warm up 2 SEAP buffer (100 μL/well) at
37 ℃.
③ Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare
the “Detection Mixture.”
④ Add 80 μL heated medium into the 96-well plate, add 120 μL Detection Mixture.
⑤ Measure absorption at 405 nm, 30 s per read for 10 reads.
⑥ Calculate enzymatic activity.
Note
SEAP levels in culture supernatants can be quantified with a pnitrophenylphosphate-based
colorimetric assay.
Protocol for Nluc assay in vitro
Material
Nano-Glo Luciferase assay kit (e.g. Promega, Catalog number selected: N1120)
Nano-Glo Luciferase assay buffer.
Nano-Glo Luciferase assay substrate.
384-well plates
Colorimetric plate-reader.
Steps
① Sample 30 μL culture medium from each well
② Add buffer and substrate into 1.5mL centrifuge tube with ratio substrate : buffer = 1:50
③ Add 7.5 μL mixture of substrate and buffer into each well of the 384-well plate, add 7.5 μL medium into each well.
④ Measure absorption
⑤ Calculate enzymatic activity.
Note
NanoLuc levels in culture supernatants characterizes the activation intensity of the downstream signaling pathway of melatonin receptor.
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