Team:NCHU Taichung/Protocol

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Description Background Design Model Result Demonstrate

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Integrated Introduction Conference Engagement Entrepreneurship

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Interlab Notebook Protocol Safety

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Protocol

Cultivation of Arabidopsis thaliana seeds

1. Use 3D Micro Shaker to shake seeds for 15 minutes with 20%/ bleach +1/1000 SDS (Tween20)
2. Remove bleach completely and add sterile water to wash three times
3. Storage at 4℃ for 4-7 days for Seed vernalization.
4. Pipette 100 μl of water and seeds to solid 1/2 M.S medium.
5. Seeds should be cultivated into plates in enough spaces to grow (Each plate should have 15-18 seeds)
6. Tilt plates and remove excess water
7. Seal the plates with parafilm
8. Put plates to growth chamber at constant temperature of 25 ± 2 ° C with 16/8 h light and dark cycle

Inoculation

1. take 8-day plants
2. choose healthy plants which have complete roots
3. Holding plant hypocotyls with tweezers and put plants in the cover of plate
4. Add proper amount bacteria of OD600=0.4 to cover whole roots and sink 1minutes.
5. Wash excess bacteria by sterile water for 1-2 second then put plants into new solid M.S medium. (For the test of how long the bacteria could stay in plants and the test of phenotype after inoculating)
6. Seal the plates with parafilm
7. For endophytic test, do further experiment

Endophytic test

1. sterilized tools: tweezers(label A、B)、dd $H_2O$、Glass Bead、Disposable pestle
2. prepared three plates of sterile water(label 1、2、3) and a plate of 10%bleach and a plate of 75% ethanol.
3. Before picking plants, sterile tweezers by alcohol burner or fuego basic then wait it to cool down.
4. Put 3 inoculated plants into 10% bleach at the same time, timing 1 minutes
   Use tweezer A to take plants out the put back to 75% ethanol, timing 30 seconds
   Use tweezer A to take plants out the put back to sterile water1, timing 30 seconds
   Use tweezer B to take plants out the put back to sterile water2, timing 30 seconds
   Use tweezer B to take plants out the put back to sterile water3, timing 30 seconds
   Use tweezer B to take plants out the put to labeled eppendorf
   Repeat above-mentioned steps for different OD600 but the same species.
   Take 100ul of sterile water3 to LB medium as the control group
5. renew sterile water1、2、3、ethanol and bleach for different bacteria species experiment
6. After grinding plants by disposable pestle, use 200ul pipette to drop 100ul to LB medium
7. Pour glass beads then shake horizontally to make fluid spread
8. Pour glass beads out to waste bottle.
9. Growth bacteria in proper temperature and wait 12-16 hours.

Cloning (3A and Gibson Assembly)

1. PCR

   KOD plus polymerase	1 μl
   dNTP	2 μl
   10XBuffer	2 μl
   Forward Primer	1 μl
   Reverse Primer	1 μl
   $MgSO_4$	2 μl
   DNA	1 μl
   $H_2O$	10 μl
2. Restriction Enzyme Digestion

   Restriction enzyme : EcoRI , XbaI , SpeI , PStI

   NEB restriction enzyme	(1μl enzyme can react with 1 μg DNA)
   DNA
   10x Cutsmart buffer	2 μl
   Add $H_2O$	to 20 μl
3. Gel extraction THERMO Gel extraction
   1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade
   2. Put into Eppendorf (remember to keep the gel weight in 100-200mg)
   3. Add equal volume of binding buffer , vortex
   4. Incubate it until all the gel is dissolved in the buffer
   5. Transfer all the solution to the purification column then centrifuge for 15,000rpm, 1min
   6. Discard the flow-through and add 100μl binding buffer then centrifuge for 15,000rpm, 1min
   7. Discard the flow through and add 700μl wash buffer then centrifuge for 15,000rpm, 1min
   8. Discard the flow through and repeat (7) process
   9. Discard the flow through and centrifuge for 15,000rpm, 7 min
   10. Transfer the purification column to the 1.5ml eppendorf
   11. Incubate it in 50-55℃ for 10 min
   12. Add 20μl $H_2O$ (prewarm) or Elution buffer into the center of the column and wait for 2-3 min
   13. Centrifuge for 15,000rpm, 7 min
   14. Use nanodrop to quantify the nucleic acids’ concentration
4. DNA clean up (after cutting, if the residue is in 5-8bp you can use DNA clean up) Genemark DNA clean up kit
   1. Add 3 volumes of binding buffer, mix completely
   2. Transfer the solution into purification column, centrifuge for 1 min in top speed
   3. Discard the flow-through and add 700μl of wash solution, centrifuge for 1 min in top speed
   4. Discard the flow-through and repeat step (3)
   5. Centrifuge for 7 min in top speed
   6. Incubate in 50-55℃
   7. Add rewarmed $H_2O$ or elution buffer into the center of the column, wait for 2-3 min
   8. Centrifuge for 7 min in top speed
   9. Use nanodrop to quantify the nucleic acids’ concentration
5. Ligation Promega T4 ligase
   1. Calcμlate the vector and insert ‘s ratio $${ng\ of\ vector\times\ kb\ size\ of\ insert\times molar\ ratio\ of\ insert\over kb\ size\ of\ vector\ vector} = ng\ of\ insert$$ vector : insert = 1 : 3 is quite optimal
   2. Add 10X buffer
   3. Add T4 ligase
   4. Add $H_2O$ till 20μl in total volume
   5. Put it in 4℃ overnight or 22℃ for 3 hr
6. Transformation Use DH5alpha E.coli comercial competent cell
   1. Add 10μl DNA into 40 μl competent cell
   2. Put it on the ice for 20-30min
   3. 42℃ heat shock
   4. Put it back onto the ice for 3 min
   5. Add 250 SOC medium and 37℃ incubate for 1 hr
   6. Plate all of the transformation and equally distribute it onto a 10 cm LB agar plate containing the appropriate antibiotic
   7. 37℃ incubate overnight
7. Gibson assembly
   1. 2–3 Fragment Assembly DNA 0.02-0.5 pmols, Gibson Assembly Master Mix 10μl, Add $H_2O$ to 20μl in total volume
   2. Incubate in 50℃ for 15 min when 2-3 fragment assembly, store on ice or in -20℃ for transformation

Protein Preparation and Extraction

1. overnight culture of recombinant strains was inoculated into 500 mL of fresh LB medium at 37°C, 200 rpm
2. Until the optical density at 600 nm was 0.8 to 1.0, 1 mM IPTG was added, and the culture was allowed to grow for another 3 to 12 h.
3. 500 mL cells were centrifuged (11,000 x g, 4 °C and 15 min).
4. The pellet was washed twice with (50mM Tris-Hcl , 0.3M Nacl pH 7.4), and centrifuged at 10000 x g, 4 °C for 10 min.
5. The cells were then re-suspended in 30 mL of the same buffer, and maintained at 0 °C for sonication.
6. Unbroken cells and cell wall materials were removed by centrifugation at 22000 x g, 4°C for 30 min
7. the supernatant was decanted and kept at 4 °C.

protein purification(Immobilized Metal Affinity Chromatography)

1. use the supernatant from protein preparation step for binding with cobalt column for 20 min
2. wash with wash buffer (50Mm Tris-Hcl, 0.3M Nacl pH7.4)
3. elut with elution buffer (50Mm Tris-Hcl, 0.3M Nacl , 0.5M imidazole pH7.4 )
4. run SDS-Page to check protein purity

Medium

1. Solid M.S medium:
   4.4g/L MS Murashige and skoog Basal Medium (store at 4° C fridge), 1.5 % Sucrose, and 0.8% agar, use HCl or KOH to adjusted pH to 5.7. autoclaved at 120°C for 20 mins.

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