Team:Shanghai HS United/Notebook
Purpose:
Have a detailed understanding to the equipment, the chemical, and the principle
What we do?
- (1)Place the equipment in to the High-Pressure Steam Sterilization Pot
- (2)Set the temperature to 121 Celsius
- (3)Make sure there are enough water in the Pot
- (4)Close the cap and start the sterilization
| Liquid LB broth | Solid LB broth |
| -NaCl 5g | -NaCl 5g |
| -Peptone 5g | -Peptone 5g |
| -Yeast extraction 2.5g | -Yeast extraction 2.5g |
| -Water 500ml | -Agar 12g |
| -Water 500mll |
(1) Reaction system:
| -Water 28 µL | -Buffer 10µL |
| -Template 5µL | -dNTP 4µLv |
| -Primer-F 1µL | -Primer-R 1µL |
| -DNA polymerase 1µ |
(2) Place the mixture in PCR
Principle of PCR:
(Stage 1): -The separation of DNA double helix-95˚C
(Stage 2): -The combination of template and primer-60˚C
-The synthesis of DNA-72˚C
(Stage 3): -The terminate of DNA amplification-16˚C
| Liquid LB broth | Solid LB broth |
| -NaCl 5g | -NaCl 5g |
| -Peptone 5g | -Peptone 5g |
| -Yeast extraction 2.5g | -Yeast extraction 2.5g |
| -Water 500ml | -Agar 12g |
| -Water 500mll |
(1)Preparation of gel:
1˚ Mix 50ml of 5X TAE to 0.75g of Agarose
2˚ Heat the mixture until transparent
3˚ Cooling for 5 minutes, and add 5µL gel red
4˚ Pour it in the gel model, and place the comb on it
5˚ Wait the gel until dry and take it off
(2)Place the gel in electrophoresis apparatus
(3)Add 1X TAE until gel is submerged in the TAE
(4)Mix the PCR production with loading buffer and drop the mixture into the hole of gel
(5)Make sure that the DNA run from cathodes to anode, so that our gel is in the right direction
(6)Start gel electrophoresis in 120v, and wait for about 20 minutes.
(7)Put the gel under the UV light, and observe it
(1)Add our original PCR production into microfuge tube
(2)Add buffer PCR-A 15µL in microfuge tube
(3)Place the microfuge tube in the centrifuge tube.
(4)Place the centrifuge tube and microfuge tube together in the centrifuge, and start centrifuge with revolving speed of 12000 rotation/minutes for 1 minute.
(5)Pour the waste in centrifuge tube
(6)Add 400µL of buffer W2, place it in centrifuge, and repeat the method again
(7)Place it in the centrifuge again without add any buffer
(8)Add 30µL water (65˚C)
(9)Use spectrophotometer to measure the concentration of our DNA solution
(1)Mix our plasmid DNA with E. coli DH10B
(2)Place the mixture on the ice
(3)Wait for 30 minutes and start heat shock (90 seconds, 42˚C)
(4)Place the mixture immediately on the ice for 2 minutes
(5)Add the mixture into liquid LB broth, and place it on incubator (37˚C) for 45 minutes
(1)Remove the extra liquid LB broth from the tube of DNA
(2)Mix the remain, and it on the solid LB broth
(3)Blent the liquid on LB broth until dry
Purpose:
Investigating the difference between LAMP amplification and PCR amplification
(1) Reaction system:
| -Water 28 µL | -Buffer 10µL |
| -Template 5µL | -dNTP 4µL |
| -Primer-F 1µL | -Primer-R 1µL |
| -DNA polymerase 1µL |
(2) Place the mixture in PCR
(1)Reaction system:
-Water 4.6µL -Template 2µL
-Mixture of primer 2µL -dUTP 1.4µL
-mixture of buffer, DNA polymerase, and dNTP 10µL
(2) Place the mixture in the water bath (65˚C) for 50 minutes (that condition is only a trial)
(3) Place our mixture in 85˚C water when times up
(1)Add 2ml liquid LB broth in each of 2 test tubes
(2)Use micropipette select two transformational E. coli, and put one in each test tube
Purpose:
Storage our target DNA, and try different condition for LAMP amplification
(1)Calculate the amount of carrier DNA and fragment DNA we need. The total amount should be 7µL
(2)Add 2µL buffer, and 1µL enzyme
(3)Place the mixture in the 37˚C water bath for 30 minutes
(1)Place them in the centrifuge
(2)Pour away the supernatant liquid, and add 250µL of Buffer S1 in the precipitate.
(3)Place the mixture in the centrifuge, and pour away the supernatant liquid. Add Buffer S2, and wait for about 5 minutes
(4)Add 350µL Buffer S3 in the mixture, and put the mixture in the centrifuge for 10 minutes.
(5). Recycle the DNA and measure the concentration
(1)Prepare different concentration gradient of our template DNA solution (from 10���-2 to 10-9)
(2)Do Lamp amplification, and analyze result by using gel electrolysis
The result of gel electrolysis shows that there is no stripe in the concentration gradient of 10-7 to 10-8, but there is a stripe in the concentration gradient of 10-9 and control group (one without template) As a result, there must be a contamination of unrelated DNA
Purpose: Investigating how Cas12a work in our gene engineering, and using test paper to test whether our product of LAMP amplification is positive or negative
(1)Prepare 3 tubes. Label one with water, one with the template without stripe, and one with template with stripe
(2)Reaction system:
-Water 14.5 µL -10X buffer 2µL
-FnCas12a enzyme 0.5µL -crRNA 0.5µL
-FITC 2µL
-Template with stripe/ Template without stripe/Water 5µL
(3) Place the test paper in each tube
As the result shown, the tube with template with stripe show a positive result on 14NT test paper, but there is a test paper show a false positive result. As a result, we should add UDG to prevent it
Purpose：
-Figure out whether primer 4 (a kind of primer mixture) is the best primer for LAMP amplification or primer 5.
-Figure out the least concentration gradient for a LAMP amplification to operate.
(1)Conditions:
Temperature: 65˚C, 67˚C
Time: 30 minutes, 50 minutes
Type of primer: Primer 4, Primer 5
(2)Gel electrolysis
According to the gel electrolysis we found that all groups with primer 4 had a stripe in the condition of 65 ° 30mins and 67 ° 50mins, while only one stripe with primer 5 was appeared. It can be proved that primer 4 is more suitable for our LAMP amplification.
(1)Conditions:
Concentration gradients: 10-3, 10-4,10-5,10-6,10-7,10-8,10-9
(2)Gel electrolysis
Purpose:
Improve the experiment from previous day by adding some new conditions
1.LAMP amplification for swine blood
(1)New conditions:
Temperature: 63˚C, 65˚C
(2)Gel electrolysis
Purpose:
Using test paper to detect the African swine fever in real blood of swine
1.LAMP amplification for swine blood (the same experiment to the last time)
