Experiments

CAFA: experiment protocols

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Culture medium:

1. LB medium:

Peptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L . After dissolving in water, autoclaved at 121C for 15min.

(If antibiotics are needed: add 1/1000 of the volume of antibiotic solution when the temperature is below 60℃)

2. LB medium (solid):

Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L and agar 15g/L. After dissolving in water, autoclaved at 121℃ for 15min.

(If antibiotics are needed: add 1/1000 of the volume of antibiotic solution when the temperature is below 60℃)

3. M9 medium

Antibiotics

1. Chloramphenicol

Weight 510 mg anhydrous chloramphenicol and dissolve it in 15mL pure ethanol, filter and sterilize it to obtain 1000X solution, which should be stored at -20℃ after separation.

2. Ampicilin

Weight 500 mg ampicilin in 5ml pure water, filter and sterilize it to obtain 1000X solution, which should be stored away from light at -20℃.

Basic operation

1. Plasmid extraction

Note: The following information is based on the instructions of TIANGENR Rapid Plasmid Extraction Kit.
1. Collection of bacteria: Take 1-4 mL of bacterial solution into 2 mL EP tube, centrifuge at 12000 RPM for 1 min, and discard the supernatant.
2. Resuspended thallus: Add 150 uL of P1 solution to the thallus precipitation (with RNaseA, purple), blown with pipette or resuspended with vortex oscillator.
3. Lyse thalli: Add 150 uL of P2 solution and mix gently upside down 6-8 times until the solution turns a slightly transparent rose red.
4. Neutralizing and alkaline: add 350 uL P5 solution, immediately and gently invert the mixture 6-8 times, until the light yellow flocculent is completely formed.
5. Separation of cell debris and macromolecular impurities: centrifuge at 12000 RPM for 2min to fully settle flocculent.
6. Assemble the adsorption column: Install the adsorption column CP3 on the collecting tube and mark the number on the cover of the adsorption column tube.
7. Plasmid DNA collection by adsorption column: Transfer the supernatant obtained in Step 5 to the assembled adsorption column, centrifuge at 12000 RPM for 30-60 s, and discard the filtrate.
8. Rinse: Add 300 uL of PWT(ethanol must be added before use) to the adsorption column, centrifuge at 12000 RPM for 30-60s, and discard the filtrate.
9. Removal of ethanol ①: Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2 min, and try to remove the bleaching liquid.
10. Remove ethanol ②: Put the adsorption column into a clean 1.5mLEP tube (mark on the tube cover), place at room temperature (or metal bath 50C) for 5-10 min, make ethanol volatilization thoroughly, to prevent residual ethanol from affecting the next experiment.
11. Solubilizing plasmid DNA: Add 30-50 uL ddH2O to the silica gel membrane in the middle of the adsorption column (ddHO at 50° C has a higher recovery rate), and let stand at room temperature for 1 min.
12. Plasmid DNA collection: centrifuge at 12000 RPM for 1 min. The filtrate in 1.5mL EP tube is the obtained plasmid DNA solution, and the adsorption column can be discarded.

2. Agarose gel electrophoresis

1. Put the rubber mold on a horizontal workbench surface, and put the rubber plate (divided into 25mL/ 50ml / 100ml) into the corresponding rubber mold. Note that there should be no air and debris between the bottom of the rubber mold and the bottom of the rubber plate, and then insert the sample comb to ensure that the bottom edge of the comb teeth will not contact with the rubber mold, otherwise it will lead to leakage of liquid in the sample holes.
2. Weigh 0.25g /0.5g/1.0g agarose into a triangular bottle, add 1×TAE buffer (25ml / 50ml / 100ml) to make 1% gel, heat in microwave oven on high heat for 0.5-1min, during which you can observe and shake well (boiling several times during the process) until the liquid in the bottle is clear and homogeneous.
3. Add the dye SYBR Safe DNA Gel Stain (10000×) 2.5ul / 5UL / 10ul, shake and mix well, carefully pour into the glue making plate (to avoid bubbles), and let stand at room temperature for 20-60 min. 4. After the solidification is complete, gently pull out the sample comb with both hands, and then form the spot sample holes separated from each other.
5. Put the gel together with the glue making plate into the electrophoresis tank and pour 1×TAE buffer until the gel surface is immersed 2-3 mm, and gently move the glue making plate and gel to eliminate bubbles in the spot sample Wells.
6. Slowly add the sample into the spot sample hole with the pipette, paying attention to avoid overflow and contamination of adjacent samples.

3. DNA recovery from agarose gel

The following information is based on the TIANGEN RUniversal DNA Purification and Recovery Kit instructions.
1. Cutting glue: Take a clean 1.5ml EP tube, mark the number on the tube cover, and add the strip containing glue block cut with a rust-free blade. 2 sol: to glue block to add 1~3 times the volume of solution PC(such as gel weight 0.1g, the volume can be regarded as 100 uL, then add 100 uL solution PC), 50 ℃ metal bath placed about 10 min, from time to time turn EP tube, to ensure that the glue block fully dissolved.
3. Assemble the adsorption column and column balance: put the adsorption column CB2 into the collection tube, add 500 uL of equilibrated BL to the adsorption column, centrifuge at 12000rpm for 1 min, and discard the filtrate. A balanced adsorption column can only be used on the same day.
4. DNA collection by adsorption column: Add the solution obtained in Step 3 to the adsorption column, place it at room temperature for 2min, centrifuge it at 12000 RPM for 30-60 s, and discard the filtrate. If the sample is larger than the adsorption column volume (800 uL), batch addition can be performed.
5. Rinse: Add 600 uL of PW to the adsorption column (ethanol should be confirmed before use), centrifuge at 12000 RPM for 30-60s, and discard the waste liquid. 6. Repeat Step 6.
7. Removal of ethanol ①: Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2 min, and try to remove the bleaching liquid.
8. Remove ethanol ②: Put the adsorption column into a clean 1.5ml EP tube, mark it on the EP tube, room temperature (or metal bath) 50℃) for 1-3 min to make ethanol volatilize.
9. Dissolve DNA: Add 30-50 uL ddHzO to the silica gel membrane in the middle of the adsorption column (ddHzO at 50° C has a higher recovery rate), and let stand at room temperature for 1 min. 1200 RPM centrifugal
After 1min, the DNA solution was collected.

4. Chemical transformation

1. Remove 50 uL of competent E. coli cells and thaw them slowly on ice.
2. Add the DNA to be transformed (plasmid or ligation/assembly product), gently mix, and place in the ice bath for 20 min.
3.After 60 seconds of heat shock at 42° C, quickly ice bath for 2 min. Do not shake EP tube during this process.
4. 300 uL of antibiotic free LB liquid medium was added and resuscitated in a shaker at 37℃ for 60 min.
5. For plasmid transformation (also known as "positive transformation", the amount of plasmid DNA added in the transformation can be >5ng), 100 uL of resuscitate bacterial solution was taken, coated on solid plates containing antibiotics, and incubated upside down at 37℃ for about 16 h.
6. For the transformation of the connection/assembly products, the resuspended bacterial solution should be centrifuged at 4000 RPM for 1 min, and 250 uL of the supernatant should be aspirated, and the remaining supernatant medium should be resuspended with the bacteria precipitation, and then all of them should be coated on the solid plate containing antibiotics, and the culture should be inverted at 37° C for about 16 h.

5. Colony identification by PCR

1. Prepare a PCR reaction system according to the table on the following page, and blow with a pipette Suction or briefly oscillate with a vortex oscillator to mix the system. When multiple colonies need to be identified at the same time, they can be combined and prepared and then separated into PCR tubes:

2. Prepare fresh LB solid medium plates containing antibiotics, and mark and number the plates and PCR tubes.
3. In the ultra-clean working table, single colonies on the transformation plate were selected with sterilized toothpicks, a line was drawn on the LB plate as a copy of the original colonies, and then immersed into the corresponding PCR tube containing PCR system. After several rotations of the toothpicks, the toothpicks were removed and thrown into the waste liquid tank.
4. Repeat the above operations to select multiple candidate single colonies.
5. Run the PCR program according to the table below:

Construct the recombined plasmid

1. T4 lysis Device and beta-galactosidase synthesis

2. Amp30E Amplification Device

3. relE characterization and reporting systems