2 × Phanta Max Buffer already contains Mg2+ at a final concentration of 2 mM.

The optimal reaction concentration varies for different templates

Set the annealing temperature according to the primer Tm value. If the primer Tm value ≥ 72°C, the annealing step can be removed and the subsequent extension step can be carried out directly (two-step PCR). If desired, a temperature gradient can be established to look for leads.

The optimal temperature for the combination of the substance and the template. In addition, the annealing temperature directly determines the amplification specificity. If poor amplification specificity is found, the annealing temperature can be appropriately increased.

Extending the extension time appropriately helps to increase the expansion yield.

Preparation of competent cells

1) Activate strains. Cultures stored at -80 °C are streaked on YPDA medium plates and incubated at 30 °C for 2-4 days.

2) Pick yeast single colonies and incubate at 3-5 mm on YPDA medium plates for 2-4 days at 30 °C.

3) When the diameter of yeast single colony reaches 2 mm, yeast cells are seeded into 3 mL of YPDA liquid medium and cultured overnight at 30°C.

4) The next day, transfer to a triangular flask containing 50 mL of YPDA liquid medium for further incubation, wait for 0D600 to reach 0.4-0.5, centrifuge at 3000 rpm for 5 min, and discard the supernatant. (Store yeast solution at 4 °C for 1 week and culture overnight with 3 L inoculation of 50 mLYPDA medium) The pellet is suspended with 25 mL of sterile deionized water. Centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend the pellet with 1.5 mL of B1 solution, centrifuge at 3,000 rpm for 5 min, and discard the supernatant.

5) Add 1 mL of B2 solution to suspension, aliquot 50 μL in 1.5 mL sterile cryopreservation tubes, and transfer libraries in 600 μL aliquots, and the competent cells are ready for transformation.

6) The prepared competent cells should be slowly frozen and then stored in a -80 °C freezer for a long time. Place the competent cells in a programmed cooling box or wrap them in a foam box with multiple layers of paper, first store in a -80 °C freezer overnight, and then remove the competent cells and place them in a -80 °C freezer for one year. Before use: Melt at room temperature and use for transformation.

Yeast plasmid transformation

1) Pipette 360 μL of the master mix(350 μLB3, 5-10 μLplasmid, add ddH2O to 360 μL) into 50 μL of competent cells and pipette the pellet repeatedly to completely suspend the yeast cells in the master mix.

2) Heat in a water bath at 30 °C for 60 min and mix every 10 min. For some species, extending the incubation time can improve conversion efficiency, but not more than 3 hours.

4) Centrifuge at 12,000 rpm at 15 sec and discard the supernatant.

5) Resuspend the pellet with 0.5 mL of YPD Plus Liquid

6) Medium and incubate at 30°C shaking for 30-60 min.

7) Centrifuge at 12,000 rpm for 15 s and discard the supernatant.

8) Resuspend the bacteria by adding 400 μL of sterile deionized water or 0.9% sodium chloride solution, coating screening medium plates, and incubating at 30°C for 2-4 days.

Aseptic operation is required throughout the transformation.

In order to ensure the conversion efficiency, it is necessary to cryoact the competent state slowly, and the competent state should not be directly frozen with liquid nitrogen.

Increasing the purity and concentration of yeast plasmids can improve the conversion efficiency.

Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 10- 15 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hr at 37°C with vigorous shaking (~300 rpm). Use a 50 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that E. coli be used for routine plasmid isolation. An OD600 reading between 2.0 and 3.0 is an indication that bacterial cells are at an optimal density for harvesting, and plasmid DNA isolation

Centrifugation at 5,000 x g for 10 minutes at room temperature.

Decant or aspirate the medium and discard.

Add 500 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.

Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page.

Transfer the cell suspension to a new 2 ml microcentrifuge tube.

Add 500 µL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.

Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.

Add 250 μL ice cold N3 Buffer. Gently invert several times until a flocculent white precipitate forms.

Note: The Buffers must be mixed throughly. If the mixture appears still viscous, brownish and conglobated, more mixing is required to completely neutralize the solution. Complete neutralization of the solution is vital of obtaining good yields.

Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.

Transfer the cleared lysate to a new 1.5 mL microcentrifuge tube. Measure the volume of the cleared lysate transferred.

Add 0.1 volume ETR Solution. Invert the tube 10 times to mix thoroughly.

Note: If you transferred 500 µL cleared lysate, then add 50 µL ETR Solution.

Incubate on ice for 10 minutes. Invert the tube serval times during the incubation.

Note: After addition of ETR Solution, the lysate should appear turbid, but it should become clear after incubation on ice.

Incubate the lysate at 42˚C for 5 minutes. The lysate should appear turbid again.

Centrifuge at 12,000 × g for 3 minutes at 25˚C. The ETR Solution will form blue layer at bottom of tube.

Transfer the top aqueous phase (cleared lysate) to a new 1.5 ml tube, add 0.5 volume absolute ethanol (room temperature, 96-100%). Gently invert 6-7 times. Incubate at room temperature for 1-2 minutes.

Insert a HiBind® DNA Mini Column II into a 2 mL Collection TubeOptional Protocol for Column Equilibration:

1) Add 100 µL 3M NaOH to the HiBind® DNA Mini Column II.

2) Centrifuge at maximum speed for 30-60 seconds.

3) Discard the filtrate and reuse the collection tube.

Transfer 700 µL mixture from Step 14 into the HiBind® DNA Mini Column II.

Centrifuge at maximum speed for 1 minute.

Discard the filtrate and reuse the collection tube.

Repeat Steps 16-18 until all of the mixture has been transferred to the column.

Add 500 µL HBC Buffer.

Note: HBC Buffer must be diluted with isopropanol before use. Please see Page 4 for instructions.

Centrifuge at maximum speed for 1 minute.

Discard the filtrate and reuse collection tube.

Add 700 µL DNA Wash Buffer.

Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 4 for instructions.

Centrifuge at maximum speed for 1 minute.

Discard the filtrate and reuse the collection tube.

Repeat Steps 23-25 for a second DNA Wash Buffer wash step.

Centrifuge the empty HiBind® DNA Mini Column II for 2 minutes at maximum speed to dry the column matrix.

Note: It is important to dry the HiBind® DNA Mini Column II matrix before elution. Residual ethanol may interfere with downstream applications.

Transfer the HiBind® DNA Mini Column II to a clean 1.5 mL microcentrifuge tube.

Add 80-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.

Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column II is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.

Let sit at room temperature for 1 minute.

Centrifuge at maximum speed for 1 minute.

Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

Store DNA at -20°C.

Use 3µl of each enzyme and scale up the reaction conditions appropriately.

The combined volume of all added enzymes should not exceed 1/10 of the total reaction volume.

If enzymes require different incubation temperature, perform sequential DNA cleavage: complete the first digestion

reaction at the lower temperature, add the second enzyme and increase the digestion temperature for the second

enzyme cleavage

Calculate the amount of DNA required for the recombinant reaction according to the formula.

To ensure the accuracy of dosing, the linearized vector can be appropriately diluted with the insert before preparing the recombinant reaction system.

Dispensing amount is not less than 1 μl.

The following reaction systems were prepared on ice:

Use a pipette to gently pipette and mix (do not shake to mix), briefly centrifuge to collect the reaction to the bottom of the tube.

Single fragment recombination reaction, 50 °C, 5 min, reduced to 4 °C or immediately placed on ice to cool.

Multi-fragment recombination reaction, 50 °C, 15 min, reduced to 4 °C or immediately placed on ice to cool.

Yeast protein extraction

1) Extraction solution preparation: Add 2ul protease inhibitor mixture to yeast protein extract B for every 500ul, mix well and put on ice for later use.

2) Take the yeast culture, centrifuge it at 4℃, 1000×g, for 5-10 minutes, carefully absorb the medium, blot it as dry as possible, and collect the yeast precipitation.

3) Wash yeast twice with cold PBS, draining as much supernatant as possible after each wash.

4) Add 250-500ul of cold yeast protein extract A to every 100-200ul volume of yeast sediment and mix thoroughly.

5) Oscillate at room temperature or 37℃ for 30 minutes to 2 hours.

6) Centrifuge at 4℃, 2000×g, for 5-10 minutes, discard the supernatant and leave the precipitation.

7) Add 300-500ul of cold yeast protein extract B to the sediment, mix well, and shake at 2-8℃ for 30-45 minutes.

8) Centrifuge at 4℃, 14000×g, for 15 min.

9) The total yeast protein can be obtained by inhaling the supernatant into another pre-cooled clean centrifuge tube.

10) The protein extract was quantified and divided into -80℃ refrigerator for reserve or directly used in downstream experiments.

Yeast protein was extracted consistent with methods described above. 100 or 200 µg of total soluble protein from each sample was mixed with 6×SDS-PAGE loading buffer and boiled (denatured) before being loaded onto10% SDSPAGE gel.

gel preparation

1) preparation: clean and assemble the glue making tank B to prepare the separation glue.

2) Equal volume mixing: take equal volume separation glue A and separation glue B and mix them well, that is, take two solutions 2/3/4ml each.

3) Add the polymerization catalyst: add 40/60/80μl of 10% ammonium persulfate solution and mix well.

4) Filling glue: Inject the mixed solution into the plastic glass plate (note: do not inject all, leave a little to judge the gel state), add appropriate amount of water or alcohol (such as isopropyl alcohol, n-butanol, etc.) to cover the lower layer of glue.

5) Preparation of concentrated glue.

6) After the separation glue solidifies, pour off the top water or alcohol.

Note: When there is a broken ray between the water (alcohol) and the glue, the gelatin has solidified.

7) Equal volume mixing: Take equal volume concentrated glue A and concentrated glue B and mix them well, that is, take 0.5/0.75/1ml of each solution.

8) Add dye to concentrated glue (optional step) : Add 1/1.5/2ul dye and mix well.(This blue dye is a small molecule dye, which will migrate along with it during electrophoresis and is located below bromophenol blue, which will not affect electrophoresis and subsequent experiments according to experimental verification)

9)Add polymerization catalyst: Then add 10/15/20μl of 10% ammonium persulfate solution and mix well.

10) Filling glue: injection into the plastic glass plate, insert comb teeth.

11) After the upper layer of glue has solidified, remove the comb teeth and use them for electrophoresis. Note: Use freshly prepared electrophoresis buffers whenever possible.

After gel separation, each gel was then blotted onto nitrocellulose membrane (Hybond™ Cytiva Life Sciences), and the target proteins were detected with specifc primary antibodies (rabbit anti-LTB) and HRP-conjugated secondary antibodies. Western band intensities, which reflect the relative amount of target proteins in the samples, were determined using the Fiji ImageJ software package.

Total RNA extraction:

1) Yeasts: Add 1ml of TROzlo reagent to 5×106yeasts.

2) The above samples are allowed to stand at 15-30 °C for 5min to fully dissociate the nucleoprotein.

3) Add 0.2ml (1 ml TROzlo reagent) chloroform, close the lid, shake vigorously for 15s and stand at 15-30°C for 2-3min.

4) Centrifuge at 12000g at 2-8°C for 15min. After centrifugation, the sample is layered, with RNA in the upper aqueous phase and protein and DNA in the lower organic phase.

5) Take the supernatant, add 0.5ml of isopropanol, mix gently, and stand at 15-30 °C for 10min, a colloidal precipitate will appear at the bottom of the tube, that is, RNA.

6) Centrifuge at 12000g at 2-8°C for 10min and discard the supernatant.

7) Add 1ml of 75% ethanol to the pellet and mix gently.

8) Centrifuge at 2-8°C 7500g for 5min and discard the supernatant.

9) Dry the RNA sample coolly (do not dry thoroughly), add an appropriate amount of DEPC water to dissolve. (can promote solubility at 55-60°C for 10min)

qPCR

Initial denaturation condition is suitable for most amplification reactions. If the template structure is complex, the initial denaturation time can be extended to 3 min to improve the initial denaturation effect.

For standard program, select 10 sec, and 3 sec can be selected for fast program.

Select 30 sec for standard program; Fast program: for amplicons within 200 bp, the shortest extension time can be set to 10 sec; for amplicons over 200 bp, the recommended extension time is 30 sec.

Different qPCR instrument types of melting curve acquisition procedures are not the same, and please select the instrument default melting curve acquisition procedures.