Medium
Luria-Bertani (LB) culture medium
For solid LB medium, an additional 1-1.5% agar powder is added and autoclaved at 121 °C for 15 min.
Yeast Extract Peptone Dextrose Medium (YPD/YEPD)
For solid YPD medium, an additional 1-1.5% agar powder is added and autoclaved at 121 °C for 15 min (glucose needs to be sterilized separately).
For yeast extract peptone galactose (YEP-Gal) medium with galactose for expression from inducible pGal promoter, galactose (20 g/L) should be used to replace anhydrous dextrose.
Yeast Synthetic Complete Medium (SC)
The mother liquor of 10×YNB and (NH4)2SO4 can be prepared and separately filtered and sterilized.
Solid SC medium with additional 15 g/L agar (glucose needs to be sterilized separately).
When preparing SC-TRP medium, CSM-TRP (0.74 g/L) should be used to replace CSM-Ura and Ura.
40% Glycerol
Autoclave at 121°C for 15 min.
Kanamycin/Streptomycin sulfate mother liquor (50 mg/mL)
The solution is sterilized by filtration, and the filtrate is divided into 1mL and stored
at -20°C. When used, add to the medium so that the final concentration is 50 μg/mL (diluted 1000 times).
Ampicillin mother liquor (100 mg/mL)
The solution is sterilized by filtration, and the filtrate is divided into 1mL and stored at -20°C.
When used, add to the medium so that the final concentration is 100 μg/mL (diluted 1000 times).
Geneticin (G418) mother liquor (20 mg/mL)
The solution is sterilized by filtration, and the filtrate is divided into 1mL and stored at -20°C.
When used, add to the medium so that the final concentration is 20 μg/mL (diluted 100 times).
1.0 M Lithium acetate solution
Autoclave at 121°C for 15 min.
ssDNA (Single-stranded carrier DNA)
Dissolved 200 mg of salmon testicle DNA in 100 mL of sterilized TE buffer (10 mM Tris-HCl, 1 mM Na2EDTA, pH 8.0).
After the salmon testicle DNA was completely dissolved in TE buffer, it was divided into 1.5 mL EP tubes and stored in the refrigerator at -20°C.
Before use, it needs to be heated in a metal bath at 100 °C for 5 minutes and then placed on ice.
Molecular Biology
Plasmid extraction
1. Take 1-5 mL of overnight cultured bacterial solution, add it to the centrifuge tube, use a conventional desktop
centrifuge, centrifuge at 12,000 rpm (~13,400×g) for 1 min, and try to remove the supernatant (if there are more bacterial
solution, the bacterial precipitate can be collected into a centrifuge tube by multiple centrifugation).
2. Add 250 μL solution P1 to the centrifuge tube where the bacterial precipitate remains (please check if
it has been added to RNase first and use pipettor or vortex oscillator to completely suspend the bacterial precipitate).
Note: If there is a bacterial block that is not thoroughly mixed, it will affect the lysis and lead to low extraction amount and purity.
3. Add 250 μL solution P2 to the centrifuge tube and gently turn it up and down for 6-8 times to fully lyse the bacteria.
Note: Mix gently, do not shake violently, so as not to break the genomic DNA and cause the extracted plasmid to be mixed with genomic
DNA fragments. At this time, the bacterial solution should be clear and viscous, and the time should not exceed 5 minutes to avoid
the destruction of the plasmid. If it does not become clear, it may be due to excessive bacteria and incomplete lysis,
and the amount of bacteria should be reduced.
4. Add 350 μL of solution P3 to the centrifuge tube, and immediately turn it gently up and down for 6-8
times to mix thoroughly. At this time, a white flocculent precipitate will appear. Centrifugation was performed
at 12,000 rpm (~13,400×g) for 10 min.
Note: P3 should be mixed immediately after addition to avoid local precipitation. If there is
still a tiny white precipitate in the supernatant, the supernatant can be removed after centrifugation again.
5. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipettor (the adsorption
column is placed in the collection tube), taking care not to suck out the precipitate as much as possible. After centrifugation at 12,000 rpm (~13,400×g) for 30-60 sec, the waste liquid in the collection tube was poured out and the adsorption column CP3 was placed into the collection tube.
6. Add 600 μL of rinse solution PW to the adsorption column CP3 (please check whether absolute ethanol has been added first),
centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.
7. Repeat step 6.
8. The adsorption column CP3 was put into the collection tube and centrifuged at 12,000 rpm (~13,400Xg) for 2 min to re
move the residual rinse solution in the adsorption column.
Note: The residual ethanol in the rinse solution will affect the subsequent enzymatic reaction (digestion, PCR, etc.) experiments. To ensure that the downstream experiments are not affected by residual ethanol, it is recommended that the adsorption column CP3 be opened and placed at room temperature for several minutes to thoroughly dry the remaining rinse solution in the adsorption material.
9. The adsorption column CP3 was placed in a clean centrifuge tube, and 50-100 μL of elution buffer EB was dropped to the middle part of the adsorption membrane, placed at room temperature for 2 min, and centrifuged at 12,000 rpm (~13,400×g) for 2 min to collect the plasmid solution into the centrifuge tube.
Note: The volume of elution buffer should not be less than 50 μL, too small volume will affect the recovery efficiency. The pH value of the eluent has a great influence on the elution efficiency. For subsequent sequencing, ddH2O should be used as the eluent, and the pH value should be within the range of 7.0-8.5. The elution efficiency will be reduced if the pH value is lower than 7.0. The DNA products should be stored at -20℃ to prevent DNA degradation. To increase the recovery of the plasmid, the resulting solution could be added back to the adsorption column, left at room temperature for 2 min, centrifuged at 12,000 rpm (~13,400×g) for 2 min, and the plasmid solution collected into a centrifuge tube.
Transforming competent Escherichia Coli DH5 α cells with plasmid DNA
1. Remove Escherichia Coli DH5α competent cells from a -80°C refrigerator and rapidly insert them into ice to dissolve.
2. Add DNA samples and mix gently. Place on ice for 30 minutes.
3. Heat the water bath at 42°C for 45 seconds, and quickly put it back on ice for about 2 minutes, taking care not to shake. 700 μl of sterile medium without antibiotics was added and mixed evenly.
4. Incubate the cells at 37°C for 1 hour with shaking (160~225 rpm), suck the appropriate volume and evenly coat into LB agar medium plates containing the corresponding antibiotics. Place the plate upside down in a 37℃ incubator for overnight culture.
PCR
(1) Prepare reaction mixture in PCR tubes:
(2) Set PCR program:
The X℃ in the annealing procedure depends on the Tm value of the primer F/R, and the X s in the extension procedure depends on the DNA product length.
DNA Digestion
Prepare reaction mixture:
Incubate the mixture at 37℃ for 1.5 hours. Then use Gel Electrophoresis and Gel Extraction to purify digestion products.
Agarose Gel Electrophoresis
(1) Agarose Gel Preparation (1 %):
1. Add the agarose and TAE buffer as this table into a flask whose volume is 2-4 times greater than the one of the solution being prepared.
2.Heat the mixture in the microwave oven for 90 seconds. After heating, remove the flask from the microwave oven with a glove and shake the flask gently to completely blend the liquid mixture. Till the solution cool down to 50-60℃, add 10 μL of DNAgreen(UV) and shake to mix. Pour the gel into a tray and insert the well comb, leave the gel to solidify for 10-15 minutes at room temperature. Once the gel is solidified, gel is ready for use.
(2) Gel Electrophoresis:
1. Remove the comb from the cast gel.
2. Place the gel into an electrophoresis system and make sure that the volume of 1×TAE buffer is sufficient.
3. After TAE submerging the wells of the gel, load DNA marker and samples.
4. Load 5-10 μL of DNA size marker.
5. Mix 3 μL of the samples with 3 μL loading buffer and then load the mixture. The volume depends on the purpose of the gel (confirmation or purification).
6. Place the electrodes with the cover over the gel system and start the electrophoresis.
7. Set current to 190 mA. Set the appropriate gel run time as 15-30 minutes. After the electrophoresis is over, switch off the system.
8. Take the gel and place it over a UV or Blue-light illuminator to visualize DNA bands for either confirmation of experimental results or gel DNA extraction.
Recovery of DNA fragments from agarose gel
1.Column equilibration step: Add 500 μl of equilibration solution BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuged at 12,000 rpm(~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. The single target DNA band was cut from the agarose gel into a clean centrifuge tube and weighed.
3. Add equal volume of solution PC to the glue block, and place it in a water bath at 50℃ for about 10 min, during which the centrifuge tube is gently turned up and down to ensure that the glue block is fully dissolved.
4. The solution obtained in the previous step was added to an adsorption column CB2, centrifuged at 12,000 rpm (~13,400×g) for 1 min, the waste liquid in the collection tube was poured out, and the adsorption column CB2 was put into the collection tube.
5. Add 600 μl of rinse solution PW to the adsorption column CB2, centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
6. Repeat step 5.
7. Put the adsorption column CB2 into the collection tube and centrifuge at 12,000 rpm(~13,400×g) for 2 min to remove the rinse solution as far as possible. The adsorption column was left at room temperature for several minutes and allowed to dry thoroughly.
8. Put the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of elution buffer EB to the middle position of the adsorption film, and place it at room temperature for 2 minutes. The DNA solution was collected by centrifugation at 12,000 rpm(~13,400×g) for 2 min.
Recovery of DNA from PCR reaction solution or enzyme digestion reaction solution:
1. Column equilibration step: Add 500 μl of equilibrium solution BL to the adsorption column CB2, centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Estimate the volume of PCR reaction solution or enzyme digestion reaction solution, add an equal volume of solution PC to it, and mix thoroughly.
3. The solution obtained in the previous step was added to an adsorption column CB2, left at room temperature for 2 min, centrifuged at 12,000 rpm(~13,400×g) for 1 min, the waste liquid in the collection tube was poured out, and the adsorption column was put into the collection tube.
4. Add 600 μl of rinse solution PW to the adsorption column CB2, centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.
5. Repeat step 4.
6. Put the adsorption column CB2 back into the collection tube and centrifuge at 12,000 rpm(~13,400×g) for 2 min to remove the rinse solution as far as possible. The adsorption column CB2 was left at room temperature for several minutes and allowed to dry thoroughly.
7. Put the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of elution buffer EB to the middle position of the adsorption film by hanging drops, and place it at room temperature for 2 min. The DNA solution was collected by centrifugation at 12,000 rpm(~13,400×g) for 2 min.
Preparation of DNA extract from the bacterial system
MightyPrep reagent for DNA is purchased from Takara.
(1) Preparation of DNA extract from the colony:
1. Add 100 µL MightyPrep reagent for DNA to a 1.5 ml Microtube.
2. Pick the colonies from the culture plate with the tip of a sterilized micropipette gun and suspended in 1 100 µL MightyPrep reagent for DNA prepared.
3. Heat at 95℃ for 10 minutes.
4. Centrifugation at 12,000 ~ 15,000 rpm for 2 minutes.
5. The supernatant was used as a template for the PCR reaction.
(2) Preparation of DNA extract from liquid culture medium:
1. After centrifugation of 1.3 ml of cell culture medium at 1000 rpm for 1 min, 1.0-1.2 ml of supernatant was recovered to a new Tube. (This step removes impurities)
2. Centrifugation at 12,000 to 15,000 rpm for 3 minutes, and discard the supernatant.
3. Add 100 µL of MightyPrep reagent for DNA and mix with an oscillator.
4. Heat at 95℃ for 10 minutes.
5. Centrifugation at 12,000 ~ 15,000 rpm for 2 minutes.
6. The supernatant was used as a template for the PCR reaction.
Gibson Assembly:
1.Prepare linearized vector by reverse PCR.
2.Design the primers and amplify the insert segments. Perform a gel extraction to purify samples.
3. Prepare Seamless Cloning reaction mixture:
The reaction system was placed in a metal bath or water bath at 50℃ for 50-60 min, and then directly transformed or stored at -20℃after cooling on ice.
Donor concentration by ethanol precipitation
1.Transfer the PCR product to a 1.5 ml centrifuge tube.
2.The volume of PCR system to be precipitated was recorded as 0.9×, in which 0.1× 3M sodium acetate solution was added, thoroughly mixed, then 2× pre-cooled absolute ethanol was added, and thoroughly mixed again.
3. Place the mixture in the refrigerator at -20°C for 15-30 min, and then centrifuge at 12000 rpm for another 10 min.
4. Gently suck off the supernatant, add 800 μL 75% ethanol along the tube wall, and slowly rotate to wash the tube wall.
5. Centrifuge at 12000 rpm for 2min, gently absorb the supernatant, and volatilize the remaining ethanol at room temperature.
6. Add appropriate amount of water to it, redissolve and precipitate to obtain DNA.
Lithium acetate transformation method of Saccharomyces cerevisiae
Protein expression and purification
Inducing expression of yeast surface display protein and antigen-antibody binding detection
Measurement of protein-REEs binding by microplate reader
Measurement of rare earth concentration based on Arsenazo III assay