-A gene of interest can be amplified from the genome of any animal, plant, bacterial plasmid containing the gene of interest as a template for PCR
-mix at each step
-template: bacteria (plasmid containing)
Fabrication of agarose gels
-DNA is negatively charged and will be electrophoresed from negative to the positive pole
-The nucleic acid dye will bind to the DNA and after beating the blue light the fluorescence (appears red)
-Marker: usually drops on the extreme edge of the lattice
Marker is a mixture of double-stranded DNA standards of different lengths, by which the DNA length of the sample can be known by comparison
-Loading: to settle the sample, glycerol containing may increase the sample density so that sedimentation into the spotting wells prevents samples from fluctuating out
Recovery of DNA
Take a clean scalpel to excise the DNA fragment per 100 mg
Determine the weight of the gel slice and transfer it 50 °C to a clean tube.
Add Buffer B to agarose gel.
Incubate the sample at 50 °C until the gel slice is completely dissolved.
Transfer the solution into the adsorption column and centrifuge 8000g for 30 seconds. Drain the liquid from the collection tube.
Add 500 μL Wash solution, centrifuge 9000g for 30 seconds, and pour ou the liquid in the collection tube.
Repeat step 6 once.
Centrifuge the empty adsorption column at 9000g for 1 min
Place the adsorption column into a new 1.5 mL micro centrifuge tube. Add 15 µL Elution Buffer and incubate at room temperature (25°C) for 1 min.
Centrifuge for 1 minute at 9000 g
Nucleic acid detection
-Detection of DNA concentration
-Experimental operation steps:
The bacteria were removed by centrifugation from the medium, and the underlying liquid was decanted
After decanting, the remaining medium was blotted onto filter paper and blotted
Keeping bacteria: keep the remaining bacteria for later experiments
Add reagents to the kit, and buffer 123 in that order (ingredients mainly ions, glucose, etc.)
The most important of buffer 1 is that there are RNases
Shake the tube and allow the buffer to mix with the enzyme
Note be sure to mix completely, without directly affecting subsequent operations
Lyase 250 μ L
Buffer 2: alkaline solution (SDS and NaOH)
SDH: disruption of cell membrane
Lysis, disruption of cell membrane / cell structure of bacteria
Denaturation, opening the duplex structure to single stranded
Mix by inversion (solution consists of turbidity → clear)
Add buffer 3
Ingredients: CH3COOH, CH3COOK
Action: neutralization, alkaline solution, renaturation (single stranded → double stranded)
Mix by shaking gently (6-8 times)
All that remains is a few flocculent impurities (cellular debris)
Centrifuge at 120000 g for 10 min to obtain the supernatant (750 mL with the goal of avoiding the pellet, change the tip of the gun every time)
Centrifuge the supernatant and pour off the following solution again
Wash by adding 500 μL wash solution
Centrifuge (1 min at 10000 rpm), take the solid
Repeat step 8.9
Centrifuge empty column(2 min at 10000 rpm)
Action: remove 75% ethanol remaining from wash solution
Allow to stand at room temperature for 2 min
Put the column into a clean 1.5mL tube, add 15μL EB buffer and centrifuge for 1min.
Put it in 37 ℃ for 1h
If the digestion time is too long, there will be non-specific cutting, which may be cut into several sections
LB Culture Medium Preparation
Yeast extract 5 g/L
Tryptone 10 g/L
NaCl 10 g/L
Agar* 1.5% of total mass
ddH2O Desired volume
*Agar is only necessary when LB culture medium is prepared as solid agar gel.
Sterilize at 121 °C for 30 minutes.
2. KANA antibiotics
Concentration of KANA antibiotics in solution is 50 mg/μL.
Functional concentration of KANA antibiotics is 50 μg/μL.
-The plasmid after ligation was aspirated and placed into competent cells
Because competent cells are very fragile, they need to be cryopreserved and manipulated gently
1. Place on ice competent cells with the plasmid for 30min Make sufficient contact of competent cells with the plasmid 2.42℃ heat shock Allow competent cells to crack, allowing plasmids to enter the cell 3. Place on ice for 2 min Allowing the bacteria to regain activity 4. Add 1 mL LB liquid medium without resistance Repair resistance (let resistance genes express) 5. The plates were incubated at 37 ° C for 1 h 6. Centrifugation Fix the bacteria to the bottom
1. Place on ice competent cells with the plasmid for 30min
Make sufficient contact of competent cells with the plasmid
2.42℃ heat shock
Allow competent cells to crack, allowing plasmids to enter the cell
3. Place on ice for 2 min
Allowing the bacteria to regain activity
4. Add 1 mL LB liquid medium without resistance
Repair resistance (let resistance genes express)
5. The plates were incubated at 37 ° C for 1 h
Fix the bacteria to the bottom
-Principle: take the bacteria as the template and use the primer amplification (PCR) of the target gene. Only the successfully connected recombinant plasmid can be copied
-Experimental operation steps:
1. - Pick up monoclonal colonies on the plate, inoculate in 10 mL LB liquid medium, and cultivate in incubator for 12 hours to 16 hours at 250 rpm.
2. - Add 500μL P1 to the CP3 adsorption column, centrifugalize at 12000 rpm for 1 minute to activate the silicon membrane.
3. - Take 1mL bacterial liquid. Centrifugate for 1 minute at 12000 rpm, and remove the supernatant.
4. - Add 250μL P1 and resuspend the precipitate.
5. - Add 250μL P2 and gently overturn the tube for 6 to 8 times.
6. - Add 350μL P3. Gently overturn the tube for 6 to 8 times, and centrifugate for 10 minutes at 12000 rpm after the white precipitates form.
7. - Collect the supernatant to the column. Centrifugate for 30 to 60 seconds at 12000 rpm, and remove the liquid.
8. - Add 600μL PW. Centrifugate for 30 to 60 seconds at 12000 rpm. Remove the liquid, and repeat the process.
9. - Centrifugate at 12000 rpm for 2 minutes.
10. - Put the column into a new centrifuge tube, add 100μL EB. Keep the tube in room temperature for 2 minutes, and centrifugate at 12000 rpm for 2 minutes.
Reaction solution preparation
-NaNO3(1 mol/L) diluted 100 times →10 mmol/L
-Di(dithioperoxo)sulfuricacid/tetrathiosulfonate（0.1 mol/L）diluted 40 times → 2.5mmol/L
-Composition of each tube configured:
5ml medium + 5μL sodium nitrate + 125 μL sodium tetrathiosulfonate
-Place the reagents needed for detection onto the microplate reader
The signal intensity has a direct relationship with the volume of liquid
-The solution was divided into 5 parts at 0 h, 1 h, 2 h, 3 h, 4 h
-The 0 h solution was replaced by blank medium, nitrate, sodium tetrathiosulfonate (because it would not be detected by the microplate reader)
-The solution was divided into 5 parts,
either of the two kind of salt above was added
only NaNO3 was added
only tetrathiosulfonate was added
both sodium nitrate and sodium tetrasulfide sulfonate were added
-Light irradiation (high energy, blue) at 485 nm wavelength was used
-Emit long wavelength light (low energy, green)