Team:NPU-China/Protocols

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DNA gel extraction

1. Excise the agarose gel slice, transfer the gel slice into a 1.5ml microfuge tube.
2. Add a 3X sample volume of Buffer DE-A. the weight of gel is equivalent to a 100ul volume.
3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 65℃ until the gel is completely dissolved(no more than 10 min)
4. Add 0.5X Buffer DE-A volume of Buffer DE-B, mix.
5. Place a miniprep colume into a 2ml microfuge tube. Transfer the solubilized agarose from step 4 into the column. Centrifuge at 12,000xg for 1 min.
6. Discard the filtrate. Add 500ul of Buffer W1. Centrifuge at 12,000xg for 30s.
7. Discard the filtrate. Add 700ul of Buffer W2. Centrifuge at 12,000xg for 30s.
8. Repeat wash with W2. Centrifuge at 12,000xg for 1min.
9. Discard the filtrate. Centrifuge at 12,000xg for 1min.
10. Transfer the miniprep column into a clean 1.5ml microfuge tube. Add 25-30ul of Eluent or deionized water to the center of the membrane. Let it stand for 1min at room temperature. Centrifuge at 12,000xg for 1min. (pre-warming the Eluent at 65℃)

Electrophoresis

Agarose-Electrophoresis is used in order to see if the PCR product is correct and seperate DNA by the number of base pairs
1. marker was the 2 kb Plus DNA Ladder
2. fill pockets with 5 µl DNA ladder or 10 µl sample volume with 6x loading buffer
3. running conditions: 130 V for 30-40 minutes

Two-step Fusion PCR

The first step:
General PCR program, 20 cycles
The second step:
General PCR program, 35 cycles

CPEC

1. Prepare the fragment premix（25uL，2X）
Since the actual addition amount of each fragment (20uL system) is often small, a 2X fragment premix of 50uL system is prepared to avoid the experimental influence caused by the addition error, whose effect usually leads to serious consequences.
2. Prepare CPEC reaction solution（20uL，1X）
10uL（Prim Star）PCR Buffer mix + 10uL fragment premix.
3. CPEC program（Take the Prim Star enzyme system as an example）
Conversion 4uL reaction solution +50uL competent state

E.coli Plasmid Extraction

1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37°C with vigorous shaking (~ 220 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation.
2. Centrifuge at 10,000 x g for 1 minute at room temperature.
3. Decant or aspirate and discard the culture media.
4. Add 250 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.
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
6. Add 250 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.
7. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.
Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.
8. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
9. Insert a DNA Mini Column into a 2 mL Collection Tube.
Optional Protocol for Column Equilibration:
1. Add 100 µL 3M NaOH to the DNA Mini Column.
2. Centrifuge at maximum speed for 30-60 seconds.
3. Discard the filtrate and reuse the collection tube.
10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the DNA Mini Column.
11. Centrifuge at maximum speed for 1 minute.
12. Discard the filtrate and reuse the collection tube.
13. Add 500 µL HBC Buffer.
Note: HBC Buffer must be diluted with 100% isopropanol before use.
14. Centrifuge at maximum speed for 1 minute.
15. Discard the filtrate and reuse collection tube.
16. Add 700 µL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use.
17. Centrifuge at maximum speed for 1 minute.
18. Discard the filtrate and reuse the collection tube.
Optional: Repeat Steps 16-18 for a second DNA Wash Buffer wash step.
19. Centrifuge the empty DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
Note: It is important to dry the DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.
20. Transfer the DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
21. Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
Note: The efficiency of eluting DNA from the DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.
22. Let sit at room temperature for 1 minute.
23. 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.
24. Store DNA at -20°C.

Gibson assembly

1. Set up the reaction.
V(ul)=(0.02*bp)/concentration(ng/ul)
2. Add the fragments into Gibson system.
3. Incubate samples in a thermocycler at 50 °C for 1h.
4. Purify the product using DNA purification kit.
5. Transform the product into the competent cells of E.coli BL21, following the transformation protocol.

Plasmid transformation

1. Pipette 100µl of competent cells and 5µl of plasmid into 1.5ml tube
2. incubate on ice 30min
2. Heat shock tubes at 42°C for 45s
3. Incubate on ice for 2min
4. Pipette 700µl LB media to each transformation
5. 37°C, 220rpm for 1h
6. Plating
7. Pick single colonies

Preparation of rho0 cells

The W303-1B monoclonal on the YPD solid plate was picked and transferred to 5 mL of fresh YPD liquid medium, cultured overnight at 30°C and 220 rpm.
50 μL of the overnight culture solution was added to 950 μL of ddH2O, then transferred to a 1cm pathlength cuvette to measure the OD600 value with a spectrophotometer.
Add an appropriate amount of the bacterial liquid, which was measured by the method mentioned above, to 5 mL of YPD liquid medium containing 5 mM ethidium bromide to a final OD600 value of 0.2, cultured at 30 °C and 220 rpm for 24 hours in the dark.
After serial passage for 1 week, a small amount of yeast liquid containing ethidium bromide was taken, streaked on YPD solid medium, and cultured in a 30°C incubator to grow a monoclonal.
The monoclonal was placed in 5 mL fresh YPD liquid medium and incubated at 30 °C and 220 rpm overnight. On the next day, collected the cells and washed them with sterile water for 2-3 times. Resuspended the cells in sterile water in 1 mL of sterile water and measured the OD600 value, which was later transferred to YPG liquid medium at an initial OD value of 0.2, sampled every two hours, and the OD600 value was simultaneously measured.

Yeast DNA Extraction

Yeast DNA Kit Protocol - Centrifugation Protocol ：
Before Starting:

• Prepare the SE Buffer, lyticase solution, and DNA Wash Buffer according to the instructions in the Preparing Reagents.
• Set the water baths and/or incubators to 30°C and 65°C.
• Set the shaking water bath to 55°C.
• Heat the Elution Buffer to 65°C.

1. Culture yeast in YPD medium to an OD600 of 10.
2. Centrifuge ≤3 mL culture (< 2 x 107) at 4,000 x g for 10 minutes.
3. Aspirate and discard the medium.
4. Resuspend cells in 480 μL SE Buffer and 40 μL lyticase solution.
Note: β-mercaptoethanol must be added to the SE Buffer before preparing the lyticase solution.
5. Incubate at 30°C for at least 30 minutes.
6. Centrifuge at 500 x g for 10 minutes to pellet the spheroblasts.
7. Resuspend cells in 200 μL TL Buffer.
8. Add 50 mg Glass Beads L. Vortex for 5 minutes.
9. Add 20 μL Proteinase K Solution. Vortex to mix well.
10. Incubate at 55°C in a shaking water bath to completely lysis the cells.
Note: Usually no more than 1 hour is required for lysis. If a shaking water bath is not available, incubate and shake or briefly vortex the samples every 20-30 minutes.
11. Add 5 μL RNase A. Invert several times to mix.
12. Let sit at room temperature for 5 minutes.
Optional: Centrifuge at 10,000 x g for 5 minutes to pellet insoluble debris. Carefully aspirate the supernatant and transfer to a clean microcentrifuge tube leaving behind any insoluble pellet.
13. Add 220 μL BL Buffer. Vortex at maximum speed for 15 seconds.
Note: A wispy precipitate may form upon addition of BL Buffer; it does not interfere with DNA recovery.
14. Incubate at 65°C for 10 minutes.
15. Add 220 μL 100% ethanol. Vortex at maximum speed for 20 seconds. If any precipitates can be seen at this point, break the precipitates by pipetting up and down 10 times.
16. Insert a DNA Mini Column into a 2 mL Collection Tube.
Optional Protocol for Column Equilibration:
1. Add 100 µL 3M NaOH to the DNA Mini Column.
2. Centrifuge at maximum speed for 30-60 seconds.
3. Discard the filtrate and reuse the collection tube.
17. Transfer the entire sample from Step 15 to the column, including any precipitates that may have formed.
18. Centrifuge at 10,000 x g for 1 minute.
19. Discard the filtrate and the Collection Tube.
20. Insert the DNA Mini Column into a new 2 mL Collection Tube.
21. Add 500 μL HBC Buffer.
Note: HBC Buffer must be diluted with isopropanol before use.
22. Centrifuge at 10,000 x g for 1 minute.
23. Discard the filtrate and reuse the Collection Tube.
24. Add 700 μL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol according to the instructions in the Preparing Reagents.
25. Centrifuge at 10,000 x g for 1 minute.
26. Discard the filtrate and reuse the Collection Tube.
27. Repeat Steps 24-26 for a second DNA Wash Buffer wash step.
28. Centrifuge the empty DNA Mini Column for 2 minutes at maximum speed (≥ 10,000 x g) to dry the column matrix.
Note: It is important to dry the column membrane before elution. Residual ethanol may interfere with downstream applications.
29. Transfer the DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube.
30. Add 50-100 μL Elution Buffer heated to 65°C.
31. Let sit at room temperature for 3 to 5 minutes.
Note: Incubating the DNA Mini Column at 65°C rather than room temperature will give a modest increase in DNA yield per elution.
32. Centrifuge at 10,000 x g for 1 minute.
33. Repeat Steps 30-32 for a second elution step.

Note: Any combination of the following steps can be used to help increase DNA yield.
• After adding the Elution Buffer, incubate the column for 5 minutes.
• Increase the elution volume.
• Repeat the elution step with fresh Elution Buffer (this may increase the yield, but decrease the concentration).
• Repeat the elution step using the eluate from the first elution (this may increase yield while maintaining elution volume).

34. Store DNA at -20°C.

Yeast Plasmid extraction

1. Inoculate 5 mL YDP medium in a 10-20 mL culture tube with yeast carrying desired plasmid and grow at 30°C with agitation for 16-24 hours.
2. Centrifuge 1-3 mL yeast culture at 5,000 × g for 5 minutes. For best results use less than 2 x 107 cells.
3. Aspirate and discard medium.
4. Add 480 μL SE Buffer/2-mercaptoethanol and 40 μL lyticase solution. Vortex at maximum speed for 1 minute. Complete resuspension of the cell pellet is vital of obtaining good yield.
Note: 2-mercaptoethanol must be added to SE Buffer before use. This mixture can be made and stored at room temperature for 1 week.
5. Incubate at 30°C for at least 30 minutes.
6. Centrifuge at 4,000 x g for 5 minutes at room temperature.
7. Aspirate and discard the supernatant.
8. Add 250 μL Solution I.
Note: RNase A must be added to Solution I before use.
9. Add 50 mg Glass Beads L. Vortex at maximum speed for 5 minutes.
10. Let sit to allow the beads to settle. Transfer the supernatant to a new 1.5 mL microcentrifuge tube (not supplied).
11. Add 250 μL Solution II. Invert and rotate the tube 4-6 times to obtain a cleared lysate.
Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. A 5 minute incubation at room temperature may be necessary. 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.
12. Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate forms.
13. Centrifuge at ≥10,000 × g for 10 minutes at room temperature.
14. Insert a DNA Mini Column into a 2 mL Collection Tube.
Optional Protocol for Column Equilibration:
1. Add 100 µL 3M NaOH to the DNA Mini Column.
2. Centrifuge at maximum speed for 30-60 seconds.
3. Discard the filtrate and reuse the collection tube.
15. Transfer 700 µL cleared lysate from Step 13 by CAREFULLY aspirating it into the DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the DNA Mini Column.
16. Centrifuge at maximum speed for 1 minute.
17. Discard the filtrate and reuse the collection tube.
18. Repeat Steps 15-17 until all cleared lysate has been transferred to the DNA Mini Column.
19. Add 500 µL HBC Buffer.
Note: HBC Buffer must be diluted with 100% isopropanol before use.
20. Centrifuge at maximum speed for 1 minute.
21. Discard the filtrate and reuse collection tube.
22. Add 700 µL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use.
23. Centrifuge at maximum speed for 1 minute.
24. Discard the filtrate and reuse the collection tube.
Optional: Repeat Steps 22-24 for a second DNA Wash Buffer wash step.
25. Centrifuge the empty DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
Note: It is important to dry the DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.
26. Transfer the DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
27. Add 50-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
Note: The efficiency of eluting DNA from the DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.
28. Let sit at room temperature for 1 minute.
29. 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.
30. Store DNA at -20°C.

Standard Yeast Transformation Protocol

1. Inoculate a single colony of the desired yeast strain to be transformed into 5 ml YPD or selective media and grow overnight at the appropriate temperature.
2. From the above overnight culture, inoculate into 5 ml YPD/selective media the appropriate amount to make the culture's optical density at 600 nm (OD600) equal to 0.1.
3. Shake at the appropriate temperature until OD600 is between 0.6 and 1.0 (Normally I wont let the OD reach over 1.0. Depends on strains, it may take 6-8 hours).
4. Pellet cells 5 min at 3,000 rpm at room temperature.
5. Wash the cells with 1ml ddH2O and transfer them into 1.5ml eppendorf tube. Pellet the cells at 3000rpm for 1min and remove the supernatant.
6. Wash the cells with 1 ml 0.1M LioAc/TE buffer. Pellet cells and resuspend in 100ul of 0.1M LioAc/TE.
7. Aliquot 20 ul of cell suspension into each of tube and add 2ul of plasmid DNA
8. Add 80ul of transformation solution and mix well by pipetting up and down for several times
Transformation Solution (each):
9. Incubate the plate at 30°C for 30min (can be longer but not shorter than 30min)
10. Heat shock by placing in a 42°C water bath for 15min
11. Pellet the cells by centrifuging at 3000 rpm for 1min.
12. Remove the supernatant.
13. Wash the cells with 100ul of 5mM CaCl2. Pellet the cells (3000rpm, 1min) and remove the supernatant
14. Add 100ul of ddH2O, mix well on the plate shaker.
15. Plate cells onto selective plates