Team:SCAU-China/protocols

MESEG

Protocols

From Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.
  • Make the following stock solutions:
    1. TAP salts
    NH4Cl 15.0 g
    MgSO4·7H2O 4.0 g
    CaCl2·2H2O 2.0 g

    Water to 1 liter


    2. Phosphate solution
    K2HPO4 28.8 g
    KH2PO4 14.4 g

    Water to 100 mL


    3. Hutner’s trace elements

    Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170

    For 1-liter final mix, dissolve each compound in the volume of water indicated.
    The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.

    compound amount water
    EDTA disodium salt 50 g 250 mL
    ZnSO4·7H2O 22 g 100 mL
    H3BO3 11.4 g 200 mL
    MnCl2·4H2O 5.06 g 50 mL
    CoCl2·6H2O 1.61 g
    CuSO4·5H2O 1.57 g
    (NH4)6Mo7O24·4H2O 1.10 g
    FeSO4·7H2O 4.99 g

    Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 mL hot 20% KOH solution (20 grams / 100 mL final volume). Do NOT use NaOH to adjust the pH.

    Bring the final solution to 1-liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, repeating the filtration, if necessary, until the solution is clear. Store refrigerated or frozen convenient aliquots. Some people shorten the time for formation of the precipitate by bubbling the solution with filtered air.

    If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.



To make the final medium, mix the following:
  1. 2.42 g Tris
  2. 25 mL solution #1 (salts)
  3. 0.375 mL solution #2 (phosphate)
  4. 1.0 mL solution #3 (trace elements)
  5. Water to 1 liter.
  6. For solid medium, add 15 g agar per liter.
  7. Autoclave.
  8. For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl.
Prepare cells
  1. Take the strain stored in 20% glycerol, and culture it at YPDA plate, 28℃ for 3days.
  2. Pick a fresh S. cerevisiae monoclonal from a YPDA plate and culture it in 3mL liquid YPDA medium, 28℃, 200 rpm overnight.
  3. Transfer the yeast solution in step2 to 10mL YPDA liquid at 1:10, 28℃, 200 rpm overnight. OD600 after step3 should be 4.0~6.0.
  4. Pack 1mL yeast solution reaching OD600 value into 1.5mL tube and centrifuge for 5000rcf/1min and remove supernatant.


Solution A:
  1. Before preparation, please boil DTT at 100 ℃ for 5min, then put it on ice.
  2. 400μL 50%PEG4000 (in TE) Repeated freezing at -80℃ and thawing before use.
  3. 50μL 1M DTT (DL-Dithiothreitol)
  4. 50μL 2M LiAC (in ddH2O)


Transformation:

Before preparation, please boil carrier DNA (Salmon sperm ssDNA) at 100 ℃ for 5min, then put it on ice.

  1. Add following solutions to cells after cell preparation:
      125mL solution A
      5mL carrier DNA
      0.1-1mg plasmid
  2. Votex it fully and incubate at 42℃ for 1 hour. Vibrate every ten minutes.
  3. Votex for 10min and centrifuge for 5000rcf/1min and remove supernatant.
  4. Resuspend the yeast at 200 μL 2% glucose.
  5. Apply the corresponding screening plate.
  6. Inverted and cultured the plate at 28 °C for 2~3 d.
Preparation of Chlamydomonas reinhardtii:

  1. Collect Chlamydomonas in logarithmic phase, centrifuge at 2000 rpm for 10 minutes, remove the culture medium.
  2. Wash it with ddH2O for three times, and use it immediately. Adjust the algae concentration to 1.6g/l with ddH2O.
Embedding:

  1. Take the adjusted concentration of algal solution and mix it with 2% sodium alginate + 7% PVA solution according to the volume ratio of 1:1.
  2. Place the beaker containing a certain volume of 0.1M CaCl2 solution on the magnetic stirrer with a built-in rotor to keep it in a slow stirring state.
  3. Drip the mixed solution of algae into CaCl2 solution at a uniform speed with No. 4.5 injection needle.
  4. Allow to stand at 4 °C for 12 hours. Then, pour out the solution and wash it with ddH2O three times (at this time, there are small balls with a diameter of about 1.5mm).
  5. Transfer the pellet into 4% (W/V) boric acid solution (pH 6.7 by HCl) and let it stand in a refrigerator at 4 ° C for 12 hours.

Stainless steel is generally recommended as the material of dissolution tank.The speed of the mixer is 100-200 rpm. If the stirring is weak, PVA particles will settle and tend to form blocks at the bottom of the dissolution tank, so pay attention. If the stirring is too strong, it is easy to bubble.

  1. Put PVA slowly into water. and stir constantly.
  2. After fully dispersed in water, stir and raise the temperature to 95 ℃, keep the temperature for about 60 minutes, PVA will be completely dissolved. Stir and cool to room temperature.

However, partial alcoholysis of PVA is easy to produce fast agglomerates during dissolution, so please pay attention to the following:

  1. The water temperature during feeding shall be kept below 25 ℃.
  2. The feeding speed of PVA is slightly slow.
  3. After feeding, fully stir according to the original temperature for 10~15 minutes, then start heating up, and continue stirring during heating up.
reference
http://www.kuraray.com.cn/Public/Uploads/image/product/20171116/20171116093132_47178.pdf

Method one


Agents and materials:
  1. Tap liquid medium with corresponding antibiotics (Zeocin, Paromomycin, Hygromycin B) and TAP plates with corresponding antibiotics (10μg/mL Zeocin, or 20μg/mL Paromomycin, or 10μg/mL Hygromycin B).
  2. Clean 0.4cm electric shock cup (soaked in absolute ethanol and dried in the sterile environment of hood); Before electric conversion, pay attention to setting a 16℃water bath, and advance the electric shock cup to 16 degrees (at least 15 minutes).
  3. Sterile 10% Tween-20 (filtration sterilization)
  4. Sucrose: Prepare 1M concentration, filter sterilization or high temperature and high-pressure sterilization; When in use, dilute it into tap, and the final concentration is 60mM.
  5. Starch (for cells without cell wall): Weigh 10g corn starch into a 50mL centrifuge tube, add 95% or 100% ethanol and completely resuspend corn starch and soak for at least 24 hours. 1000g, centrifuged for 1min, removed ethanol, added an appropriate amount of screening medium +60mM sucrose for resuspension, repeated this step 5 times, and finally resuspended it in screening medium +60mM sucrose at 25% (W/V)
  6. Boiled salmon sperm DNA (10mg/mL) (optional)
  7. Sterile 50mL centrifuge tubes

Protocol:
  1. Culture Chlamydomonas cells in 25mL TAP liquid medium until the cell density is about 1 ~ 5x106 cells / mL (22℃, 3 ~ 4 days, 120 rpm), then transfer an appropriate amount of Chlamydomonas cells to 100mL TAP liquid medium and continue to culture until the cell density is about 1 ~ 5x106 cells/mL;
  2. Place Chlamydomonas cells on ice for 10 min, transfer them to a 50 mL sterile centrifuge tube, and add sterile 10% Tween-20 in the ratio of 1:2000. For example, 50 μL 10% Tween-20 is added to 100 mL of cells (optional: Tween-20 needs to be added when fewer cells are obtained, which is conducive to cell redundancy)
  3. Collect cells by centrifugation: 4 ° C, 2000g, 5min (if tween-20 is added in step 2, centrifugation at 4 °C, 5000g, 5min can be selected) (This time 3000rpm, 5min).
  4. Operate in the hood: remove the supernatant as much as possible, resuspend the cells with precooled TAP (+ 60 mM sucrose) liquid medium, the cell density after resuspension is about 4x108 cells/mL, and place it on ice for 15 minutes; At the same time, precooling the electric shock cup;
  5. Operation in the hood: add 250 μL of resuspended cells, 5 μL of 10 mg/mL boiled salmon sperm DNA, and 7 μL of DNA to be transformed in a 0.4 cm electric shock cup, mix evenly, and cover;
  6. Take a water bath at 16 °C for 5 min, place it in the electromotor, and set the parameters (Bio Rad gene pulser II: 0.6 -0.65 kV, 25 μF and no resistance; BTX Gemini X2: voltage 800V, resistance 1575 9, capacitance 50 μF).
  7. Wipe off the water in the shell of the electric shock cup with absorbent paper, put it flat into the electric shock instrument, clamp the electric shock cup on both sides, and put down the safety cover; Press pulse, that is, conduct electric shock and record the time. 10-14 ms is the normal time range (the electric conversion is completed when the prompt tone is heard); Immediately after electric shock, transfer to ice and place for 10min.
  8. If resuscitation is not required, the transformed cells shall be directly coated on the screening plate (for Chlamydomonas cells with cell wall defects, 0.6 ~ 0.8mL 25% corn starch solution shall be added and coated on the surface of the plate). After the plate is dried in a sterile environment, it shall be cultured under the set culture conditions: (resuscitation is required this time, see article 9 of this section)
  9. If resuscitation is needed, transfer the transformed cells to 40mL tap + 60mm sucrose liquid medium for culture for 24-48h, and recover overnight in slow and weak light on the shaking table. The cells were collected by centrifugation, resuspended in 1mL TAP liquid medium, and coated on the plate. The method is the same as that in step 6. Treatment of electric shock Cup: clean it after use, add absolute ethanol into the electrode cup, cover it with a hat and store it for a long time. Take it out before electric conversion and dry it in hood (under sterile conditions).

Method two


Preparation
  1. Early logarithmic Chlamydomonas cc4533 (cmj030) (OD650 between 0.6-1.0)
  2. TOS (containing 40mm / L sucrose (filtration sterilization) TAP solution)
  3. Plate containing 10 ug /ml of zeocin

Protocol
  1. Well cultured Chlamydomonas, 160ml, 3500rpm (1000g), 10min
  2. Pour out the supernatant and add 10ml TOS for suspension then centrifuge at 3500rpm (1000g) for 5min
  3. Pour out the supernatant and add 10ml TOS for suspension then centrifuge at 3500rpm (1000g) for 5min
  4. Pour out the supernatant and adjust cell concentration to 2x108cells / ml.
  5. Then add the following agents into 1.5 ml centrifuge tube.
    6. Transformation group Control group
    2ug plasmid (linearized) the same volume of water
    250ul Chlamydomonas 250ul Chlamydomonas
    Mix well, 16 degrees for at least 30min (or more than 80min).
The protocol of preparing competent E. coli cells by CaCl2
  1. Take E. coli (DH-5α) cell tube from -70 ℃ and inoculate on the SOB plate without resistance at 37 ℃, 18~28 h to get single colony.
  2. Culture E. coli in 1mL liquid SOB medium each tube at 200 rpm under 37 ℃ overnight about 3~4 h.
  3. Drop 500 μL cultured E. coli solution in to 50 mL new liquid SOB medium (1:100 dilution) to culture 2~3 h until the solution reaches about OD600=0.3~0.6. Then put it on ice.
  4. Centrifuge the E. coli solution in a 2 mL tube (1.5mL/tube) at 5000 rpm, 4 ℃ for 2 min to collect the cells and drop out supernatant.
  5. Add 300~500 μL cold 0.1 M CaCl2 solution to resuspend the E. coli cells and put the tube on the ice quietly for about 25 min.
  6. Centrifuge the preparing cell tube at 5000 rpm, 4 ℃ for 2 min to collect the cells and drop out supernatant.
  7. Add 50~100 μL cold 0.1 M CaCl2 and resuspend the cells on the ice. Then store the cell at 4℃.
E. coli (DH-5α) transformation protocol
  1. Take competent cell tube from 4 ℃ and put on the ice.
  2. Add 10 μL plasmid solution into each tube of 100 μL competent cells and put it back on the ice quietly for 30 min.
  3. Use metal bath at 42 ℃ to hot shock for 90 s, then put it on ice for 5min.
  4. Add 500~800μL LB to culture at 220 rpm under 37 ℃ for 1 h to recover resistant.
  5. Centrifuge the tube at 12000 rpm for 1 min, discard the upper liquid and leave 100 microliters for heavy suspension.
  6. Put the transformed E. coli solution on the surface of the exact resistant LB media plate and place under 37 ℃ for about 10~12 h (overnight).
100mL 200mL
YNB(0.67%) 0.67g 1.34g
-ura(0.2%) 0.077g 0.154g
Galactose (2%) / glucose (2%) 2g 4g
Adjust pH = 5.8 and add ddH2O to make the corresponding volume
Agar(2%) 2g 4g
Sterilization at 121 ℃ for 15 minutes
100mL 200mL
YPD 5g 10g
Ade (Yeast specific) 0.012g 0.024g
Agar 2g 4g
Add ddH2O to make the corresponding volume.
  • A suitable concentration of agarose gel was prepared, and DNA fragments were separated by electrophoresis. After the DNA fragment was separated, the gel was placed under the ultraviolet lamp, and the gel containing DNA fragments was quickly cut off and the excess gel was removed as far as possible.
  • Weigh the gel block and transfer it to 1.5 or 2 mL centrifuge tube. According to 100mg gel block is equivalent to 100 μL. For volume calculation, add equal volume buffer GDP. 50~55 degrees C water bath for 7~10 minutes, let the gel block completely dissolve. During the water bath, mix upside down twice to accelerate the sol. If the weight of the gel block is 200mg, 200 μL Buffer GDP is added. When the gel concentration exceeds 2%, 2 times the volume of Buffer GDP was added. When processing more than 5KB fragments, after adding 3 times volume (gel volume), add 1 times volume (gel volume) isopropanol and mix it evenly. Then follow step 3 to operate.
  • The droplets on the tube wall are collected by brief centrifugation. The HiPure DNA Mini column was sheathed in a 2mL centrifuge tube. Put 700 μL transfer the solution to the column. 12000g centrifuged for 30 ~ 60 seconds.
  • (Optional: Sol more than 700 μL) Pour the waste filtrate and put the column back into a 2mL centrifuge tube. Transfer the remaining solution to the column, 12000g centrifugation for 30 ~ 60 seconds.
  • Discard the filtrate and put the column back into a 2mL centrifuge tube. Add 300 μL buffer GDP into the column. Let it stand for 1 minute. 12000g centrifuged for 30 ~ 60 seconds.
  • Discard the filtrate and put the column back into a 2mL centrifuge tube. Add 600 μL buffer DW2 (diluted with absolute ethanol) into the column, 12000g centrifugation 30 ~ 60 seconds.
  • (Optional) discard the filtrate and put the column back into a 2mL centrifuge tube. Add 600 μL buffe DW2 (diluted with absolute ethanol) into the column. 12000g centrifugation for 30 ~ 60 seconds.
  • Discard the filtrate and put the column back into a 2mL centrifuge tube, 12000g centrifuge for 2 minutes. For some sensitive applications (when most of the eluent needs to be added to the connecting reaction solution): open the cover of the column and dry by air for 5 minutes to completely remove ethanol.
  • The column is sleeved in a 1.5mL centrifuge tube and 15-30 μL elusion buffer is added to the center of column membrane. Let it stand for 2min, then, 12000g centrifuged for 1 minute. Drop the post and save the DNA at -20℃.

Gel recover kit is from Guangzhou Magen Biotechnology.

  • Reaction system preparation:
    10μL
    2X Taq Master Mix(Vazyme,Dye plus) 5μL
    Primer F 0.5μL
    Primer R 0.5μL
    ddH2O 3μL
    Bacterial solution to be tested 1μL
  • PCR program

    Pre denaturation 95 ℃ for 3min
    Denaturation: 95 ℃ 15s
    Renaturation 55 ℃ 15s
    Extension 72 ℃ 30s
    Number of repetitions X34
    Extend again at 72 ℃ for 5min

  • 1.5% agarose (1.5g / 100mL) + 0.5X TBE 100mL.
  • Put it in the microwave for melting.
  • Accession 6 μ L/100mL EB
  • Pour the mixture into the mold and wait for freezing.
  • Point (DL2000 DNA marker) 5 μL,10X DNA Loading buffer(Vazyme)1 μL mixed with DNA sample.
  • Electrophoresis (3~5V/cm)
  • The reaction system is prepared according to the following table.
    10μL 50μL
    ScaI enzyme 0.2μL 1μL
    10X Buffer 1μL 5μL
    plasmid DNA 2μL 10μL
    Nuclease free ddH2O To 6.8μL To 50μL
  • Incubate at 37 ° C for one hour.

The enzyme was bought from NEB company.

Agents:
  • Sealing solution: 5% non-fat milk powder, prepared by 1XPBS).
  • Transfer buffer (1L): Tris 5.8g, Glycine 2.9g, Methanol 200mL(add before use).
  • 10XPBS: 0.2mol/L Tris-HCl (pH 7.5), 1.5 mol/L NaCl.
  • 1XPBST: 1XPBS, 0.1%TritonX-100 (1000μL)
Protocol:
  • Electrophoresis and membrane transfer
    1. x μL sample n μL protein extract + 2 × protein sample buffer.
    2. Constant temperature at 100 ℃ and denaturation for 10min.
    3. Loading, then electrophoresis at 8mA, 120min.
    4. Transfer to NC (Nitrocellulose) membrane: cut off the excess gel, assemble the film according to the gel on the negative electrode (black) and the film on the positive electrode (white), and no bubbles are allowed. There are two layers of filter paper (soaked in transfer buffer) between the sponge pad and gel or NC membrane.. The membrane needs to be activated using ethanol for one minute. The filter paper needs to be soaked in the transfer buffer.
    5. Constant current 80mA, overnight.
  • Seal off and crossbreeding
    1. Sealing: transfer the membrane into the sealing solution, 37 ℃, 50rpm and incubate the membrane at room temperature rinsing, 70rpm for 1h.then discard sealing solution
    2. Add hybrid solution I (the first antibody), 70rpm, 1h.
    3. 1 × PBST wash 3 times, 10min each time.
    4. Add hybrid solution II (the second antibody), room temperature, 70rpm, 1h.
    5. 1 × PBST wash for 3 times, 10min each time.
  • Development
    1. Transfer the washed membrane to a clean container then add luminous solution A and B, 500 μL each.
    2. Install the film in the X-ray cassette (watch out for bubbles) then use X-ray film to develop in a certain time (dark room).
    3. Develop in developing solution then wash in water. Finally, fixing in fixing solution (dark room).
Agents:
  • 30% gel storage solution: Acrylamide 30g, Bis-acrylamide 0.8g, 100mL ddH2O.
  • 10% ammonium persulfate (AP) solution: AP 2g, 20mL ddH2O.
  • 1 mol/L HCl
  • Constant volume to 100 mL.
  • Concentration gel buffer, 0.5mol/l Tris – HCl: Tris 6g, pH 6.8, adjust pH value to 6.8 by HCl. Constant volume to 100 mL.
  • 10%SDS: SDS 5g, dissolved in 50mL hot water.
  • 10%TEMED: prepare 20ml when use.
  • 3Xelectrode buffer: Tris 6g, Glycine 28.8g, SDS 1g. Constant volume to 1000 mL.
Protocol:
  • Clean and dry the glass plate with a blower.
  • Install electrophoresis tank and glass plate.
  • Prepare four 100ml small beakers for water, sealing solution, concentrated glue and separating glue respectively. Prepare 1 straw and 1 glass rod respectively.
  • Use a small beaker to prepare the sealing glue solution according to the table below, mix it evenly, pour it into the sealing groove of the electrophoresis tank immediately, and let it stand for 5 ~ 10 min until it solidifies.
    Agents Sealing gel/μL 12% Separation gel/μL 5% Concentration gel/μL
    30% gel storage solution 2000 4000 830
    Concentration gel buffer (pH 6.8) - - 2500
    Separation gel buffer (pH8.8) - 2500 -
    water 6000 3200 1480
    10% SDS - 100 50
    10% TEMED 200 100 70
    10% AP 120 100 70
    Total 8320 10000 5000
  • Fill a small beaker with water, fill the two layers of glass with water, and check whether there is water leakage at the bottom and both sides of the glass. If there is water leakage, reinstall it.
  • Use a small beaker to prepare the lower layer of gel (separation gel) according to the above table. The gel concentration is 12%, mix evenly, fill the gel, its height is 1 ~ 1.5 cm away from the comb, and then cover a layer of water with a straw. The setting time is 15 ~ 30 min. When the gel and water are layered, pour out the water.
  • Use a small beaker to prepare the upper gel (concentrated gel) according to above table. The gel concentration is 5%. Mix it evenly, fill it with glue and insert it into the comb. The setting time is 10 ~ 20 min. Pull out the comb after solidification.
  • Add diluted electrode buffer into the upper tank of the electrophoresis tank and soak it to the low glass surface, but it shall not exceed the high glass surface. Pay attention not to water leakage.
  • Add diluted electrode buffer into the lower tank of the electrophoresis tank, and the volume is basically the same as that of the upper tank.
  • Loading and then setting electrophoresis program according to your plan.
  • Select a single colony and inoculate it in YPDA liquid medium until OD600 =1.
  • The cell suspension was diluted to OD600=0.5 and OD600=0.01, respectively.
  • Then use a pipette to suck 1-5μL (depending on the size of the plate) and drop it vertically. Repeat 3 times for each concentration.
  • Centrifuge at 10000g for 1 minute. Collect 1-5mL cell.
  • Discard the medium. Gently tap on the absorbent paper to absorb the residual liquid. Add 250 μL buffer P1 / RNase A mixture to resuspend the cells in a high-speed vortex.
  • Add 250 μL buffer NP2 into the heavy suspension and mix it upside down gently for 8-10 times.
  • Add 350 μl buffer NP3 and immediately reverse it 8-10 times to completely neutralize the solution.
  • Centrifuge at 13000 – 16000 for 2 minutes.
  • Install HiPure DNA Mini column II in the collection tube. Carefully transfer the supernatant to the column and centrifuge at 13000g for 30-60 minutes.
  • Discard the filtrate. Put the column into the collection tube. Add 500 μL buffer PW1 to the column and centrifuge at 13000g for 30-60 seconds.
  • Discard the filtrate. Put the column into the collection tube. Add 600 μL buffer PW2 (diluted with absolute ethanol) to the column and centrifuge at 13000g for 30-60 seconds.
  • (Optional) repeat step 8 one time.
  • Discard the filtrate. Put the column into the collection tube. Centrifuge at 13000g for 3min.
  • Put the column in a sterilized 1.5ml centrifuge tube. Add 30 - 100 μL elusion buffer or sterilized ddH2O to the center of the membrane of the column, stand for 1 minute, 13000g centrifuge for 1 minute to elute DNA.
  • Store at -20℃.