Straw after crushing processing, select 48 straw powder, placed in plastic bags, with filter paper and rope will straw powder packaging, with the concentration of 95% ethanol and round bottom flask extraction, until the extract faded yellow, extraction after straw powder, placed in an open box, drying test moisture in straw powder.
1. Take 18.837 g straw powder, add 1% diluted sulfuric acid (1.925 mL concentrated 98% + 350 mL deionized water), and react in a 121°C sterilization pot for 1 hour;
2. After the reaction, a cloth funnel was used for solid-liquid separation with two layers of filter paper to remove the liquid and retain the solid on the cloth funnel. The solid is first cleaned with absolute ethanol to pH is neutral, and then cleaned with heated deionized water until the color of the cleaning liquid is clarified. Solids were collected into plastic bags, and the moisture content of the solids was measured after an overnight test.
Add cellulase to the solid, and a buffer of pH4.8 (HAc-NaAc). During enzymatic digestion, the substrate concentration is 10%, the enzyme is 25 FPU/g, the enzyme and substrate are added to the brown cone flask, placed in a shaker (50°C, 150 rpm, shake 48h), burchell funnel for solid liquid separation, liquid, the liquid contains the glucose we need. The concentration of glucose in the liquid was measured by using a glucose analyzer.
| HAc-NaAc Buffer(0.2 mol/L,18°C) | ||
| pH | 0.2 mol/L NaAc | 0.2 mol/L HAc |
| 4.8 | 5.9 mL | 4.10 mL |
(NaAc·3H2O Mr=136.09,0.2 mol/L solution is 27.22 g;0.2 mol/L HAc is 11.55 mL/L glacial acetic acid)
The laboratory used sodium anhydrous acetate with a molecular weight of 82.03
| Configure the NaAc solution | |
| 1 L system | 250 mL system |
| 16.406 g | 4.1015 g |
| Configure the HAc solution | |
| 1 L system | 250 mL system |
| 11.55 mL | 2.8875 mL |
| Absolutely dry solid | The amount of enzyme | |
| 60 mL system | 6 g | 0.9677 g |
| 25 mL system | 2.5 g | 0.4032 g |
Add moisture solid = absolutely dry solid (1% solid water)
a. Conventional PCR system
| Name | Volume( μL ) |
| Mix enzyme | 50 |
| primer 1 | 4 |
| primer 2 | 4 |
| template | 1 |
| ddH2O | To 100 |
b. Fusion PCR system(Take the example of two fragment fusions)
| Name | Volume( μL ) |
| Mix enzyme | 50 |
| primer 1 | 4 |
| primer 2 | 4 |
| template 1,template 2 | 1 each |
| ddH2O | To 100 |
Notes
a. After the system is prepared, it needs to be shaken well.
b. Generally, 200 μL PCR tubes are used to aliquot the above system into two small tubes, each 50 μL.
| Process | Temperature(°C) | Time(s) |
| pre-denaturation | 95 | 180 |
| denaturation | 95 | 15 |
| annealling | T(m) | 15 |
| extension | 72 | t |
| final extension | 72 | 300 |
| saving | 12 | ∞ |
Notes：
a. "T(m)"is generally determined by the nature of the primer, and "t" is generally determined by the length of the gene of interest.
b. The number of cycles for denaturation, annealing, and extension is generally 35.
Optimal cloning vector usage=[0.02×Number of base pairs of cloning vector÷concentration]ng
Optimal insertion fragment usage=[0.04×Number of inserted fragment base pairs÷concentration]ng
matters need attention：The unit of concentration is ng/μL
Fragment1 usage=[0.02×Number of base pairs of cloning vector÷concentration]ng
Fragment2 usage=[0.02×Number of base pairs of cloning vector÷concentration]ng
Fragment3 usage=[0.02×Number of base pairs of cloning vector÷concentration]ng
matters need attention：The unit of concentration is ng/μL
In order to ensure the accuracy of adding samples, the content of linearized carrier and inserted fragment can be adjusted appropriately before preparing the recombination reaction system, and the adding amount of each component is not less than 1μL.
| Component | Recombination reaction |
| Linearized carrier | X μL |
| Insertion fragment | Y μL |
| 2 ×ClonExpress Mix | 5 μL |
| ddH2O | To 10 μL |
50°C,15 min. Reduce to 4°C or cool immediately on ice. The recombinant plasmid was obtained.
a. Single colonies E. coli Top10 were inoculated in 5 mL of LB liquid medium and cultivated at 37°C, 200 r / min for 15-20h;
b. Medium containing OD600≈0.05 was transferred into 100 mL of LB liquid medium, cultivated at 37°C, 200 r / min for about 2h and OD600 to 0.4~0.6;
c. The bacterial liquid was centrifuged at 8,000 r / min 4°C for 5 min to collect the bacteria, and then the supernatant was removed;
d. 20 mL of pre-cooled 100 mmol / L magnesium chloride solution was added to the tube with bacteria,gently mixed, and incubated on ice for 30 min;
e. After centrifugation at 8,000 r / min 4°C for 5 min, the bacteria were collected and discard the supernatant;
f. 1 mL of pre-cooled 200 mmol / L calcium chloride solution and 1 mL of pre-cooled 15% glycerol solution was added to the tube, gently mixed, packed 80 μL per tube, use it immediately or store it in a negative 80°C refrigerator.
a. E.coli Top10 competent cell were thawed on ice;
b. 8 μL of recombinant plasmid product was added to 100 μL of competent cells;
c. Shake well, and let it stand on ice for 30 min;
d. 900 μL LB of liquid medium (without antibiotics) was added to the tube and incubated in a 37°C shaker for 1h;
e. The resistant LB solid medium plates were prewarmed in a 37°C incubator;
f. The centrifuge tube was centrifuged at 5,000 rpm for 5 min, discarded 900 μL of supernatant, suspended with the remaining medium, coating aseptically on plates containing antibiotics;
g. The medium plates were inverted in an incubator for 37°C for 12 to 16 h.
a. After E. coli Top10 competent cells to be transformed are cultured in resistant plates for a period of time, several single colonies of uniform size grow, which can be verified by colony PCR;
b. Determine the system to be built, record the amount of Mix enzyme, upstream primer, downstream primer and dd water to be added in the notebook. And select the single colony to be picked on the back of the petri dish, and number them in order;
c. According to the data recorded in the previous step in the PCR tube to add upstream primers, downstream primers and dd water, put it in a centrifuge spin for a few seconds, to ensure that the mixture on the wall reached the bottom of the tube. Finally, add Mix enzyme (because the enzyme is easy to lose activity, so the enzyme stored in the ice box), elastic tube wall makes it mix.
a. Take out the PCR tube (already in advance of sterilization) with the same number of single colonies to be picked and put it on the centrifuge rack, and write the number on the PCR tube cover. Use the pipete gun to divide the constructed system into the PCR tube (the pipete gun should be adjusted back to the maximum range and put in after the use);
b. Open the ultra-clean working table for ultraviolet sterilization 15-20 minutes in advance, turn off the ultraviolet light, turn on the lighting and the ventilation;
c. Light the alcohol lamp, burn the tweezers with the alcohol lamp, then remove the toothpick that has sterilized the bacteria in the conical bottle in advance, pick up the right amount of bacteria in the numbered order in the plate, stir the toothpick into the PCR tube to mix it well. Finally close the ultra-clean working table after the operation is completed.
a. Prepare an agarose gel concentration of 0.8% according to the amount of glue prepared. Weigh the desired agarose powder on the balance with weighing paper, add the powder to the beaker, then add 1×TAE buffer (diluted from 50×TAE buffer) (the total liquid volume should not exceed 30% of the volume of the conical flask, avoid liquid flushing out of the topical bottle due to high temperature). Heat it in a microwave oven, take the beaker during heating and gently shake. Mix the agarose with the buffer, while avoiding liquid overflow. When the solution is clear and transparent, stop heating after naked eye observation of the non-turbidity state, rinse it with cold water to cool it slightly, then the nucleic acid dye (Gelview) is added according to the ratio of 1:10,000, m. b. After placing the board on the gel slot and inserting the comb, slowly pour the solution into the gel tank (avoiding air bubbles) until cooling and solidification;
b. After PCR amplification, open the instrument, take out the PCR tube and arrange it in the numbered order, turn off the instrument, prepare the 10 μL pipete gun and gun head, and take out the required marker from the refrigerator;
c. Remove the comb, then remove the gel and place it in an electrophoresis apparatus, and poured in 1×TAE buffer to soak the gel, set aside the first hole. From the second hole, inject the liquid in the PCR tube into the gel hole in the numbered order. Finally, 10 μL of marker was punched in the first hole, all the holes into the cover of the electrophoresis device, turn on the instrument, reckon by time. Turn off the instrument after 20-25 minutes of running. Remove the gel and put it into the side gel imaging system aside. Turn on the ultraviolet lamp for irradiation, on the computer screen for a strip generated. If a clear and correct band is observed, colony PCR is successful verification (pay attention to saving the photos in time).
A. Take the bacteria liquid cultured overnight and centrifuge it in a centrifuge for 10,000 rpm for 1 min, and discard the medium;
B. Add 500 μL of Buffer P1 to the centrifuge tube with the bacteria remaining, mix well and transfer to a 2 mL centrifuge tube;
C. Add 500 μL of Buffer P2 to the centrifuge tube where the bacteria remain, mix gentlysly, and lyse the bacteria (Solution state viscous and translucent);
D. Add 700 μL of Buffer P3 to the centrifuge tube and mix gently, at which point a white flocculent precipitate appears Centrifuge at 12,000 rpm for 10 min (the upper layer is clear overnight, and the lower layer is white flocculent precipitate);
E. Place the FastPure DNA Mini Collection adsorption column in the Collection collection tube, take only the supernatant, add it to the adsorption column, centrifuge at 12,000 rpm for 30-60 s, and remove the liquid in the Collection collection tube;
F. Add 600 μL of Buffer PW2 (already diluted with absolute ethanol) to the adsorption column at 12,000 rpm for 30-60 s, removing the liquid from the Collection collection tube;
G. Repeat step 6, add 600 μL of Buffer PW2 (already diluted with absolute ethanol), centrifuge at 12,000 rpm in the adsorption column for 30-60s, and remove the liquid from the Collection collection tube;
H. Place the FastPure DNA Mini Collection adsorption column in the Collection collection tube, centrifuge at 12,000 rpm for 60s, and completely remove the rinse solution in the adsorption column;
I. Place the FastPure DNA Mini Collection adsorption column in a new 1.5 mL centrifuge tube, add 30 μL of pure water to the center of the membrane, centrifuge at 12,000 rpm for 60s, and elute out the DNA;
J. Turn on the UV spectrophotometer, set the mode to ssDNA, and calibrate after washing the connector with pure water. Take 1 μL of sample under the probe and measure the concentration;
K. Send the obtained products to professional companies for sequencing.
a. Single-colony Corynebacterium glutamicum were inoculated in 15 mL BHIS of liquid medium and grown at 30°C, 250 rpm for 12h;
b. The activated organisms were connected to 100 mL BHIS liquid medium and controlled for starting OD≈ 0.3;
c. And in a 30°C, 250 rpm shaker for 1 h 45 min to give an OD around 1;
d. The above bacterial solution was transferred to a 50 mL centrifuge tube and centrifuged at 4°C and 4,000 rpm for 10 min before removing the supernatant;
e. 30 mL of precooled 10% glycerol resuspension was added, and the supernatant was discarded by centrifugation at 4°C at 4,000 rpm for 10 min, and the bacteria were collected;
f. Repeat step (5) twice;
g. Recool 1 mL of 10% glycerol and pack in a 1.5 mL EP tube in 100 μL of -80°C.
a. 1 μg of recombinant plasmid was added to 100 μL of Corynebacterium glutamicum competent cells, beaten and mixed. Ice bath for 5-10 min;
b. The required antibiotics were added to BHIS liquid medium by shaking. And the BHIS liquid medium was evenly divided into 10 mL centrifuge tubes in 6 mL each;
c. The recombinant plasmid and the competent mixture were added to the shock cup and clicked for each shock cup 3 times using the shock instrument;
d. After clicking, remove the mixture from the shock cup and add it to the packed 6 mL BHIS liquid medium;
e. 46°C water bath for 6 min;
f. After the water bath, the centrifuge tube was incubated in a 30°C shaker for 4h (rotation speed: 200-250 rpm);
g. After incubation, centrifuge tube for 10 min (4,000 rpm), discard the supernatant, and retain 200 μL of culture medium and mix;
h. The culture medium in 7 was coated on a BHIS solid plate. And cultured at 30°C for 36 – 48h.
a. Liquid: For every 100 mL liquid medium, 1g sodium chloride, 1g peptone, 0.5g yeast extract;
b. Solid: For every 100 mL liquid medium, 1g sodium chloride, 1g peptone, 0.5g yeast extract, 2g agarose;
c. After adding water, sterilize in vertical autoclave at 121°C for 20 minutes;
d. After sterilization, the liquid medium was placed at room temperature, and the solid medium was placed in the oven at 60°C.
a. Every 150 mL BHIS liquid medium, add 3.7g BHI and 9.1g sorbic acid, add ultra-pure water and mix well;
b. Sterilize in a high-pressure steam sterilizer at 115°C for 20 minutes;
c. After sterilization, put in room temperature for use.
Disposable gloves are used for packaging materials, sterilization temperature is 123°C, time is 20- 60 minutes. After sterilization, they were placed in the oven at 60°C for drying and ready for use.
a. Wash the used petri dish with tap water once;
b. Use ddH2O to moisten the petri dish again, so that water droplets do not hang on the petri dish;
c. Pace the washed petri dish at room temperature to dry;
d. Put the dried petri dish in a vertical high-pressure steam sterilizer to sterilize;
e. Put the sterilized petri dishes in the oven at 60°C to dry;
f. Take out the dried petri dishes and encapsulate them with newspaper, every 10 petri dishes;
g. Put the encapsulated petri dishes in the vertical high-pressure steam sterilization pot for sterilization;
h. After sterilization, take out the encapsulated petri dish and put it in the oven for use.
a. Wear disposable gloves for subpacking: put PCR tube, 1.5 mL, 2 mL and 10 mL into 300 mL conical bottle or iron box respectively, and use sealing film to seal; Put 50 mL centrifuge tube into iron box, 10 in each box. Finally use newspaper and retrograde wrap;
b. Place the encapsulated centrifugal tube in a vertical high-pressure steam sterilizer to sterilize;
c. After the end of sterilization, placed in the oven, waiting for use;
d. Use in the sterile bellows: remove the centrifugal tube from the oven, put it in the sterile bellows, UV sterilization for 20 minutes, you can carry out subsequent operations;
e. Use in the open space of the laboratory: it can be taken out directly for use without strict aseptic conditions.
a. Wear disposable gloves for operation, separate the gun head, and use newspaper to encapsulate the gun head box;
b. In the vertical high pressure steam sterilization pot;
c. After sterilization, put in the oven for use;
d. Use in the sterile bellows: remove from the oven, placed in the sterile bellows, ultraviolet sterilization for more than 20 minutes, then can be used;
e. Use in the open environment of the laboratory: after taking out of the oven, use directly.
Wear disposable gloves throughout the preparation process to prevent contamination of nucleic acid reagents
a. Configure the concentration of 0.8% agarose gel electrophoresis;
b. Clean the gel plate and gel comb with 1X solution;
c. Use analytical balance to weigh 0.8g agar-agar powder;
d. Use a special measuring cylinder to measure 100 mL of 1X solution;
e. Mix agar-agar powder and solution into conical bottle;
f. Heat the solution in the microwave oven. The solution should not boil;
g. After heating for a while, shake and heat again until the solution is clear and transparent the solution is heated to clarify the water bath cooling, add nucleic acid dye, the concentration is 0.01%, mix;
h. Insert the gel comb, pour the solution, wait for cooling after use.
