Team:AISSU Union/Design



Configure 2× FastPfu Fly Mix, according to the following system for subsequent PCR system:

The PCR system for gel-recovered fragments is as follows:

The FastPfu Fly DNA Polymerase reaction procedure is as follows:

The colony PCR system is as follows:

The reaction procedure of 2×Taq DNA Polymerase is as follows:

After the colony PCR system is set up, take 0.5 µL points of the mixed system on the corresponding antibiotic-resistant replica for subsequent inoculation of plasmids and bacterial species preservation.

Pipette Tips Recycling Steps

After configured the corresponding system of 1% of Agarose gel, adding 10,000 diluted fold nucleic acid dye SYBR Safe, and then expanding PCR product to 140V voltage for running gel, and finally fragment was retained by gel extraction kit which is selected from Beijing the gel recovery kit of Tiangen Biochemical Technology Co., Ltd. has been optimized according to experimental requirements. The specific operation following steps are:

Preparation of yellow pipette tip adsorption column CA2: Sterilize the tweezers with 75% alcohol, take out the blue fixing ring in the adsorption column CA2 of the glue recovery kit, taking out the filter membrane in the adsorption column, and use tweezers to take half of each into the yellow gun head, plug the filter membrane as tightly as possible.

1.  Column equilibration steps: put the yellow pipette tip adsorption column CA2 in a new tube, adding 200 µL balanced solution BL to it, centrifuging at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the middle of collection tube.

2.  Cut a single target DNA band from the agarose gel and put it into a 1.5 mL clean centrifuge tube.

3.  Add 150-250 µL of solution PN depending on the size of the cut gel block (if it is not clear whether the gel block is small, adding 200 µl), and place it in a metal bath at 65°C for 5-10 minutes. During this time, gently turn the centrifuge tube up and down until the gel block completely dissolved. Note: At this time, place the sterile dd H2O on a metal bath at 65°C for preheating.

Note: For the recycle of small fragments <300bp, you can add 1/2 the volume of the gel block (usually 100 µL) of isopropanol after adding PN to complete the sol to increase the DNA recovery rate; after the gel block is completely dissolved, please lower the temperature to Load the column at room temperature because the adsorption column has a strong ability to bind DNA at room temperature.

4.  Add 200 µL of the solution obtained in the previous step to the yellow pipette tip adsorption column CA2 after column equilibration (the adsorption column is placed in the collection tube), mark the yellow pipette tip, place it at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute. Pour off the waste liquid in the collection tube, and put the yellow pipette tip adsorption column CA2 back into the collection tube. Then add the remaining solution to the yellow pipette tip adsorption column CA2 without placing it, and centrifuge at 12000rpm for 1min.

5.  Add 200µL of rinsing solution PW to the yellow pipette tip adsorption column CA2 (please check whether absolute ethanol has been added before using), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the yellow pipette tip adsorption column CA2 back into the collection tube middle.

6.  Repeat step 5.

7.  Put the yellow tip adsorption column CA2 back into the collection tube, 12000 rpm air from 2min, try a divisible rinse of the PW. Take an appropriate amount of 1.5mL centrifuge tube, put the yellow pipette tip adsorption column CA2 in a new 1.5mL centrifuge tube, and place it in a metal bath at 65°C for 5 minutes to thoroughly dry the rinsing liquid PW to prevent residual rinsing liquid from affecting the next experiment.

8.  Take 65 ℃ preheated dd H2O around 16-20μL, was added dropwise to a central membrane adsorption, placing at room temperature for 2min, 12000 rpm centrifugation for 2min; the collected DNA was added back to the centrifugal adsorption column, placed 2min, 12000 rpm centrifugation for 2min, Collect DNA.

Plasmid extraction

Use the plasmid small extraction kit of Beijing Tiangen Biochemical Technology Co., Ltd. to extract plasmids from the bacteria cultured overnight. The specific operation steps are as follows:

1.  Centrifuge 5 mL of the bacterial solution cultured overnight at 4000 rpm for 8-10 min to collect the bacterial cells, and remove the supernatant as much as possible.


2.  Was added to the bacterial pellet leaving bacteria shake tube 150 µL solution Pl (previously added RNAse and TIANRed), by pipetting or vortexing (the degree of mixing of the cells on the extraction and subsequent cleavage of the plasmid Quantity and purity), and transfer it from the 14 mL shaking tube to a new 1.5 mL EP tube.


Note: The addition of TIANRed reagent has no effect on post- PCR, enzyme digestion and sequencing. After thoroughly inverting and mixing, the solution is clear red. Add the mixed solution to the collected bacteria and mix thoroughly. Due to the presence of the bacteria, the mixed solution is turbid red.

3.  Add 150 µL P2 buffer to a 1.5 mL EP tube, and gently mix upside down 7-8 times to ensure that the bacteria are fully lysed.

Note: At this time, the bacterial solution should become clear and viscous. If it does not become clear, it may be due to too much bacteria and incomplete lysis. The amount of bacteria should be reduced (do not shake vigorously, otherwise it will interrupt the genomic DNA and cause the extracted plasmid If there are DNA fragments, the operation time of this step should not exceed 5 min to avoid damage to the plasmid).

Due to the use of TIANRed, after adding solution P2 and mixing thoroughly, the color of the solution is clear purple. If there is a turbid red in the purple, it means that the lysis is not sufficient. Continue to mix until the color of the solution completely changes to a clear purple.


4. Add 350 µL solution P5 buffer to the 1.5 mL EP tube, gently invert and mix 12-20 times to ensure that the bacteria are fully mixed, at this time flocculent precipitation will appear, then immediately centrifuge at 12000 rpm for 12 min.

Note: After P5 is added, it should be inverted and mixed immediately to avoid local micro-precipitation. The supernatant containing a small amount of small white precipitate has no effect on the subsequent test. If the supernatant is still mixed with micro-precipitate, it can be centrifuged again and there will be no subsequent experiment. Influence. If there are a lot of tiny white precipitates in the supernatant, centrifuge again and take the supernatant.

Due to the use of TIANRed, after adding solution P5 and mixing thoroughly, the solution is clear yellow. If purple is mixed with yellow, it means that the renaturation is insufficient. Continue to mix until the color of the solution is completely clear yellow.

5. Use a pipette to transfer the supernatant collected in the previous step to the CP3 adsorption column (the adsorption column is placed in the collection tube), try not to absorb the precipitate. Centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put CP3 back into the collection tube.


6.Add 400 µL  of PWT washing solution to the adsorption column CP3 (confirm whether absolute ethanol is added in advance), centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put CP3 back into the collection tube.


7.Repeat the operation (6).


8. Put the CP3 back into the collection tube, and centrifuge the empty tube again at 12000 rpm for 2 minutes to ensure that the residual washing solution on the CP3 adsorption membrane is completely removed. Then let it stand for 5 min in a metal bath at 60°C to completely dry the remaining washing liquid on the adsorption membrane.


9. Put CP3 into a new clean 1.5 mL EP tube, add 50-60 µL of sterilized dd H 2 O to the center of the CP3 adsorption membrane ( preheat at 65°C in advance to improve the elution efficiency), and let stand at room temperature 2 After min , centrifuge at 12000 rpm for 2 min to collect the plasmid in the tube (if you want to improve the elution efficiency, you can aspirate the plasmid in the collection tube and add it to the center of the adsorption membrane again, and centrifuge at 12000 rpm for 2 min to enrich the plasmid again). The plasmid should be used immediately, otherwise it should be stored at -20°C to prevent DNA degradation.


Gibson Assembly

The Gibson Assembly system is as follows:

According to the above system, the configuration 10 µL of Gibson assembly system at 60℃ the reaction 1h to complete the assembly clip, and then Escherichia coli DH5α transformed.

Golden Gate Assembly

The Golden Gate Assembly system is as follows:

If the number of inserts is 1~10 , use 420 units ; if the number of inserts is more than 10 , use 630units; Available endonuclease: BsmBI-v2(NEB #R0739, 10U/µl), BsaI-HFv2 ( NEB #R3733S 20U/µl); If the number of inserts is 1~10 , use 24 units ; if the number of inserts is greater than 10 , use 36 units;  The amount of each insert can be based on the amount of vector added and the vector and the length of the insert.

The Golden Gate Assembly reaction procedure is as follows:

According to the above reaction system and program configuration 20µL of the Golden Gate assembly system, after completion of the reaction for E. coli DH5α transformed.

The enzyme linkage system is as follows:

Table 9 T4 DNA Ligase enzyme linkage system

According to the above system, configure a 10 µL ligation product, incubate at 20°C for 4 h to complete the construction of the ligation product, and then transform E. coli DH5α.


Escherichia coli competent transformation


1. Take out the prepared competent state in the ultra-low temperature refrigerator at -80 ℃ and thaw it on ice. After the defrosting is complete, add 10 µL reaction product (add enzyme or assembly product immediately after thawing to avoid affecting the transformation quality of competent state). Flick the tube wall with fingers to mix the bacteria, and bath in ice for 20-30 min.

2. After the ice bath is completed, heat shock in a 42 ℃ water bath for 45-90 seconds, and immediately let it stand on ice for 5 min .

3. Add 400 µL SOC liquid medium and resuscitate at 220 rpm in a shaker at 37 ℃ for 1 h.

4. Spread all the recovered bacterial liquid on the corresponding resistant LB solid medium, and invert it overnight in an incubator at 37 °C.


Yeast Competent Preparation

1.  Spread the stored yeast on YPD solid medium and incubate at 30 °C for 2 days . Pick a single colony was inoculated into YPD liquid medium at 30 shaker in ℃ 230rpm cultures 18-20 h .

2. Re-inoculate the overnight cultured yeast into 10mL YPD medium so that the initial OD600 is 0.1-0.15; culture it in a shaker at 30°C, 230 rpm to OD600 = 0.4~0.6, and collect the bacteria by centrifugation at 4000 rpm for 5 min body;

3. Resuspend with 1mL sterile water, centrifuge at 4000rpm, 5min to remove the supernatant; add 1mL cell suspension (TE/LiAc/H2O=1:1:8) at 4000rpm, 5min, centrifuge again to remove the supernatant;

4. Gently resuspend the bacteria with 150 µL cell suspension to prepare yeast competent cells.


Yeast assembly and transformation

1. Boil the single-stranded salmon sperm DNA (10mg/mL) in a boiling water bath for 10 minutes before use, and then use an ice bath for later use. Add 20 µL  to the 300 µL conversion solution (TE/ LiAc/ PEG3350 =1:1:8) Mix gently.

2. Take the purified DNA fragments and ensure that the final concentration of the added fragments is ≈200 ng, then the final concentration of the linearized vector is ≈50 ng (4: 1), and the fragments are in an equimolar ratio. Mix, add to the yeast competent cells, flick and mix (to ensure optimal transformation efficiency, the total volume of the fragment mixture should not exceed 60 µL, within this transformation volume range, the concentration of the mixture can be expanded to ensure higher Assembly efficiency).

3. Add the prepared yeast transformation solution to the yeast competent cells (the yeast transformation solution is configured according to the number of transformations), and vortex at low frequency to mix.

4. Incubate at 30°C (water bath, metal bath or 30°C incubator) for 45 min.

5. Then place it in a 42 ℃ water bath for 15 minutes, ice bath for 2 minutes, and centrifuge at 4000 rpm for 5 minutes to remove the supernatant, resuspend and wash the bacteria with 600µL ddH 2 O, then centrifuge at 4000 rpm for 5 minutes to remove the supernatant again, and add 120µL ddH 2 O to resuspend the bacteria, spread on the SC-U auxotrophic solid plate, incubate at 30 ℃ for 3 days, select recombinants for verification  



1. YPD medium ( 1L ) : 10g yeast extract, 20g peptone dissolved in 900 mL deionized water at 121 ℃ for 20 minutes , add 100mL 20% glucose mother liquor; add 15g agar powder before sterilization, it is a solid agar medium.

2. 20% glucose mother liquor: Weigh 20g glucose in 100mL deionized water, sterilize it at 115 ℃ for 15 minutes , and store it at 4 ℃ for later use .

3. SC-Ura Defective Medium ( 1L ) : Weigh 6.7g Yeast Synthetic Drop-out Medium without uracil , take 1.3g Yeast Synthetic Drop-out Medium without uracil , dissolve in 900mL deionized water, sterilize at 121 ℃ for 20 minutes , add 100 mL 20% glucose or galactose mother liquor is used as the carbon source; 15g agar powder is added before sterilization to form a solid agar medium.

4. 10 × LiAc solution: Weigh 8.16g LiAc • 2H 2 O solid, dissolve it in 80 mL deionized water, adjust the pH to 7.5 with HAc, add deionized water to make the volume to 100 mL, obtain 0.8M LiAc solution and quench Bacteria spare.

5. 10 × TE solution: 1.211g Tris, 0.37g EDTA dissolved in 80 mL deionized water, adjust the pH to 7.5 with HCl, add deionized water to make the volume to 100 mL, obtain 0.1M Tris-HCl, 10mM EDTA sterilization spare.

6. 50% PEG3350: Weigh 25g of PEG3350 solid and dilute to 50ml with deionized water, filter and sterilize 0.22 µm for use.


Yeast Recombinant Screening

After the yeast colony grows to a certain size (2 mm~3 mm), pick the yeast transformants into 10 µL sterilized dd H2O water and mix well, stir and mix, and take 4 µL dots in the auxotrophic type of SC-Ura On the medium plate, the adapter plate style is shown in the figure below, and the colony PCR is initially screened after overnight culture at 30°C.

Due to the thicker cell walls of Saccharomyces cerevisiae, it is necessary to break the walls before doing the yeast colony PCR. Use a pipette tip to dip the yeast colony and resuspend in 10µL 20mM sterile NaOH, and rupture the yeast cell wall in a PCR machine according to the procedure in Table 10:

Take out the processed bacterial solution from the PCR machine, mix it and take 1 µL as a template, use Taq DNA Polymerase to verify the bacterial solution PCR according to the PCR reaction system in Table 11 and the PCR program in Table 5, and screen the correct yeast recombinant: