Team:NTHU Taiwan/Experiment
Protocol
Cloning
Normal PCR/ Gradient PCR (find best annealing temperature)
1. Prepare PCR mixture (units: μl)
| Item | number |
|---|---|
| DNA template | 1 |
| Taq polymerase | 0.5 |
| dNTP(10mM) | 1 |
| Foeward primer(20μM) | 1 |
| Reverse primer(20μM) | 1 |
| 10×PCR buffer | 5 |
| ddH2O | 40.5 |
DNA amount: 100ng
2. Program
| number | time |
|---|---|
| 95 | preheat |
| 95 | 5 min |
| 95 | 30s |
| 55 | 30s |
| 72 | 30s go back to step3 repeat for 30 cycles |
| 72 | 5 min |
| 4 |
1. Transfer reaction product to a 1.5 ml microcentrifuge tube. Add 5 volumes of Gel/PCR Buffer to the sample then vortex.(*If the mixture has turned from yellow to purple, add 10 µl of 3M sodium acetate (pH5.0) and mix thoroughly.)
2. Transfer the sample mixture to the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through.
3. Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the DFH Column and let it stand for 1 minute. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Centrifuge again at 14-16,000 x g for 3 minutes to dry the column matrix.
4. Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 20-50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.
5. If the final concentration of purified DNA is too low (lower than 15ng/μl), we would put the elution buffer (in our protocol is ddH2O in 55°c) with purified DNA back to the column and stand for 10 minutes to elute the remained DNA.
1. Set up the following reaction in a microcentrifuge tube on ice. (100μl reaction)
| Item | volume |
|---|---|
| DNA (depends on concentration) | 1μg |
| Cut Smart buffer (10X) | 10μl |
| Enzyme 1 | 1μl |
| Enzyme 2 | 1μl |
2. Digest for 1 hour
3. Use NEB double digest finder (https://nebcloner.neb.com/#!/redigest)to find the best condition for digestion.
4. Concentration of restriction enzyme should be lower than 5% to avoid star activity.
As mentioned in PCR clean up
1. Set up the following reaction in a microcentrifuge tube on ice. (20μl reaction)
| Item | volume |
|---|---|
| T4 DNA ligase | 1μl |
| T4 DNA ligase buffer (10X) | 1lμ |
| Insert sequence | |
| Backbone | 100ng |
| ddH2O | To 20μl |
2. Use NEBiocaculator(https://nebiocalculator.neb.com/#!/ligation)to find the ratio of backbone and insert sequence.
3. Incubate at 16°C overnight
1. Add 1-5µl containing ligation product to the 100μl of the cells.
2. Place the mixture on ice for 30 minutes.
3. Heat shock at exactly 42°C for exactly 45 seconds.
4. Place on ice for 5 minutes.
5. Repeat step 3 and 4
6. Pipette 900 µl of room temperature LB broth without antibiotic into the mixture. (for recovery)
7. Place at 37°C for 60 minutes.
8. Warm selection plates to 37°C.
9. Centrifuge at 14,000-16,000xg to remove the excess LB and resuspend the cells with 200μl of LB with antibiotic. Spread onto a selection plate and incubate for 16 hours at 37°C.
1. Pick a single colony with a 20-200μl tip.
2. Put the tip into the tube with 5ml LB broth with antibiotics.
3. Place at 37°C for 60 minutes. Shake vigorously (250 rpm).
Harvesting
Transfer 1.0-1.5 ml of cultured bacterial cells to a microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room temperature to form a cell pellet then discard the supernatant completely.
Resuspension
Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet. Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved.
Cell Lysis
Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2 acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.
Neutralization
Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.
DNA Binding
Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
Wash
Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new 1.5 ml microcentrifuge tube. Perform Wash Buffer steps twice for salt sensitive downstream applications.
Elution
Add 50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow the Elution Buffer, TE or water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.
1. Making 1.2% agarose gel (small)
| Item | volume |
|---|---|
| TBE buffer (0.5X) | 20ml |
| Agarose | 0.24g |
2. Loading sample
DNA marker: 1μl of 6X (loading dye/ Novel juice) + 5μl of DNA ladder
DNA sample: 1.5μl of 6X (loading dye/ Novel juice) + 2μl of DNA
3. Start the electrophoresis 100V for 30 minutes.
Prepare annealing mixture
| Ingredients | volume (μl) |
|---|---|
| 100μM sense oligo | 0.5 |
| 100μM antisense oligo | 0.5 |
| 10X T4 ligase buffer | 5 |
| T4 PNK | 1 |
| H2O | 43 |
- incubation at 37 degree C for 10 mins
- incubation at 95 degree C for 5 mins
- Cool down overnight
Nematode Subculture
| Ingrendients | g/L |
|---|---|
| Dextrose | 20 |
| Potato extract | 4 |
| Agar | 15 |
Performing in laminar flow.(once a week)
Prepare for the nematodes’ food
1. Scratch about 0.2cm2 mold from tubes with transferring loop
2. Transfer the mold to new PDA slant tube
Once open or close the tubes, every step need to sterilize over the fire
Nematodes grow on PDA slant tube containing A. citri.(once a week)
1. Add 3ml ddH2O to tube, rolling the slant tube to wash down the nematodes
2. Add 1300 μl of the suspension to 2 microcentrifuge tube, centrifugation at 2800g for 5 minutes
3. Remove the supernatant(1200 μl)
4. Pipetting both of the tubes, and mix them into one microcentrifuge tube
5. Get 50μl nematode suspension with P100 pipetman (in laminar flow)
6. Transfer to new agar slant tubes containing mold(in laminar flow)
7. Culture in incubator at 24 degC
Once open or close the tubes, every step need to sterilize over the fire
Callus culture
1. Remove shells first, then sink seeds in tap water for over 14 hours.
2. Sink seeds in 70% alcohol for 0.5-1 minute.
3. Wash them with ddH2O three times.
4. Sterilize by sinking in 0.1% HgCl for 10-15 minutes.
5. Wash them with ddH2O 5-8 times.
6. Take embryos from seeds in laminar flow
1. Composition of LM medium(pH: 5.8~6.2)
| Item | volume |
|---|---|
| ½ LM medium (loblolly pine medium) | |
| 2,4-dichloro-phenoxyacetic acid (2,4-D) | 2.0mg/L |
| 6-benzyladenine (6-BA) | 1.0mg/L |
| casein hydrolysate | 1000mg/L |
| L-glutamine | 500mg/L |
| inositol | 1000mg/L |
| sucrose | 30g/L |
| agarose | 5g/L |
1. Composition of MS medium
| Item | volume |
|---|---|
| Murashige and Skoog (MS) medium | |
| 2,4-dichloro-phenoxyacetic acid (2,4-D) | 10mg/L |
| 6-benzyladenine (6-BA) | 4mg/L |
| kinetin | 4mg/L |
1. Dipping embryogenic callus into bacterial suspension for 30 to 60 min
2. Transfer the callus, without rinsing, to the MS medium.
3. Co-cultivation for 2 d in the dark at 28 °C
4. Rinse the infected callus by sterile water
5. Blotted dry with sterile filter paper to remove an excess of bacteria
6. Transfer onto the selective medium for 9 - 12 weeks
*Murashige and Skoog (MS) medium containing
500 mg/L carbenicillin
10, 50, 100 or 150 mg/L kanamycin
Agobacterium Culture
| Ingredients | mg/L |
|---|---|
| Dipotassium hydrogen phosphate | 0.5 |
| Magnesium Sulfate (MgSO4-7H2O) | 0.2 |
| Sodium Chloride | 0.1 |
| Mannitol | 10 |
| Yeast Extract | 0.4 |
- Prepare YM Medium.
- Add DNA to microcentrifuge tubes.
- To determine transformation efficiency, add 1 µl of the pBI121 control DNA to a microcentrifuge tube.
- For plasmid DNA from minipreps, precipitate the reactions with ethanol and resuspend in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. The concentration of resuspended DNA should not exceed 100 ng/µl. Add 1 µl of the DNA to a microcentrifuge tube.
- Thaw the ElectroMAX A. tumefaciens LBA4404 Cells on wet ice.
- When cells are thawed, mix cells by tapping gently. Add 20 µl of cells to each chilled microcentrifuge tube containing DNA.
- Refreeze any unused cells in a dry ice/ethanol bath for 5 minutes before returning them to the -80°C freezer. Do not use liquid nitrogen.
- Pipet the cell/DNA mixture into a chilled 0.1 cm cuvette and electroporate. Using the following electroporator conditions: 1.5 kV, 200 Ω, 25 µF .
- To the cells in the cuvette, add 1.0 ml of room temperature YM Medium and transfer the solution to a 15 ml snap-cap tube.
- Shake at 225 rpm (30°C) for 3 hours.
- Dilute cells transformed with the control pBI121 DNA 1:10 with YM Medium. Spread 100 µl of this dilution on prewarmed YM plates containing 100 µg/ml streptomycin and 50 µg/ml kanamycin.
- Dilute experimental reactions as necessary and spread 100-200 µl of this dilution on prewarmed selective plates.
- Incubate plates for 48-56 hours at 30°C
Others
Storage
RNA Isolation REAGENT, chloroform: 4°C
isopropanol, linear acrylamide(5 mg/mL), 75% ethanol(in DEPC-treated water): -20°C
RNase-free water: room temperature
- Collect worms in a 1.5ml centrifuge tube and store in -80°C and wait for RNA extraction.
Homogenization
- Add approximately 0.1ml of 0.5mm zicronic beads and 400μl of RNA isolation reagent into each tube.
- Use parafilm to seal the tubes.
- Use blender (speed 1500rpm for 5 mins)
- Add 700μl RNA isolation reagent.
- Incubate the homogenized samples for 5 mins at RT.
Homogenization
- Add 250μl of chloroform.
- Vortex tubes vigorously for 15 secs and incubate them at RT for 2 to 3 mins.
- Centrifuge the samples at 12000g for 15mins at 4°C. (following centrifugation, the mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase)
Precipitation
- Transfer the aqueous phase to new tubes.
- Add 600μl of isopropanol and 1μl linear acrylamide to precipitate the RNA and invert gently
- Incubate sample at RT for 30-60 mins and centrifuge at 12000g for 15mins at 4°C.
Wash
- Remove the supernatant
- Add at least 1ml of 75% ethanol.
- Mix the sample by gently inverting and centrifuge at 12000g for 15mins for 4°C.
- Circle the RNA pellet by marker pen..
- Remove the supernatant.
Re-dissolving the RNA
- Briefly dry the RNA pellet.
- Add 5μl of nuclease free water onto the RNA pellet, and wait for a few minutes.
- Use pipetman to pipet gently to dissolve the RNA.
Preliminary quantification
- Add 1μl product and 1μl nuclease free water to a PCR tube.
- Use nanodrop to quantify RNA amount and check the 260/280, 230/260 ratio.
- Recycle the quantified samples in case of running electrophoresis.