Team Protocol
Cloning
PCR
Reaction system:
H2O 38 \(\mu\)L
10x Taq buffer 5 \(\mu\)L
2.5mM dNTP 4 \(\mu\)L
Primer 1 1 \(\mu\)L
Primer 2 1 \(\mu\)L
Template 0.5 \(\mu\)L
Taq 0.5 \(\mu\)L
Process:
\(\begin{equation}\left. \begin{array}{lcl} {94°C\ 10min} \\ {94°C\ 30s} \\{58°C\ 30s} \end{array} \right\} Cycle\ 30\end {equation}\)
94°C 10min
94°C 30s
58°C 30s
72°C 2min30s
72°C 10min
10°C ---
Electrophoresis---Gel Purification
Material:
Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain
Protocol:
We used gelstain to stain the DNA and imaged it in a Transilluminator.
We used the gel extraction kit to get the objective fragment.
Digestion
| 50 μL reaction system | |||||
|---|---|---|---|---|---|
| Reagent | 10x H buffer | EcoR I | Pat I | Plasmid | H2O |
| Dosage | 5 \(\mu\)L | 1.5 \(\mu\)L | 1.5 \(\mu\)L | 15 \(\mu\)L | 27 \(\mu\)l |
| 10 μL reaction system | |||||
|---|---|---|---|---|---|
| Reagent | 10x H buffer | EcoR I | Pat I | Plasmid | H2O |
| Dosage | 1 \(\mu\)L | 0.3 \(\mu\)L | 0.3 \(\mu\)L | 3 \(\mu\)L | 5.4 \(\mu\)L |
Ligation
| Ligation reaction system | ||||
|---|---|---|---|---|
| Reagent | Cph8 | PSB1C3 | T4 buffer | T4 ligase |
| Dosage | 14 \(\mu\)L | 3 \(\mu\)L | 2 \(\mu\)L | 1 \(\mu\)L |
Transformation
Material:
| LB liquid medium | |||
|---|---|---|---|
| Reagent | Tryptone | Yeast extract powder | NaCl |
| Dosage | 10 g/L | 5 g/L | 10 \(\mu\)L |
Protocol:
preparation of the competent cells
20\(\mu\)L ligation product + 50\(\mu\)L cells
Heatshock of E.coli BW25113(42°C,90s)
Put on ice(2min)
Add 800\(\mu\)L LB media and incubate for 1.5h(37°C, 150rpm)
Centrifuge at 4000rpm for 1min and remove 900\(\mu\)L supernatant
Resuspend the pellets using the left supernatant
Spread plates(with ampicillin)
Incubate for 12~16h(37°C)
Detetion
SDS-PAGE
Materials:
| Gel | Tris-HCl | Acr/Bis 30% | SDS 10% | ddH2O | TEMED | AP 10% |
|---|---|---|---|---|---|---|
| Stacking Gel(4%) | pH=6.8 500\(\mu\)L | 500 \(\mu\)L | 25 \(\mu\)L | 1350 \(\mu\)L | 2.5 \(\mu\)L | 12.5 \(\mu\)L |
| Running Gel(12%) | pH=8.8 1250 \(\mu\)L | 2000 \(\mu\)L | 50 \(\mu\)L | 1675 \(\mu\)L | 2.5 \(\mu\)L | 25 \(\mu\)L |
| Running Gel(18%) | pH=8.8 1250 \(\mu\)L | 3000 \(\mu\)L | 50 \(\mu\)L | 675 \(\mu\)L | 2.5 \(\mu\)L | 25 \(\mu\)L |
| Running Buffer | ||||
|---|---|---|---|---|
| Reagent | Tris-HCl | Glycine | (w/v) SDS | |
| Dosage | 25 mmol/L | 0.192 mol/L | 0.1% | |
Protocol:
The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.
After ensuring that the equipment is waterproof, the 12 % (or 18%) running gel is mixed and filled into the chamber. Pipetting about 1 ml of H2O on top of the running gel to seal the gel.
After polymerization, the remaining H2O is removed and the 12 % stacking gel is filled on top. Insert a comb to create sample pockets.
After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via the SDS gel.
After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120 V.
Detection of Baiting Nematodes
Materials:
Pure chemical compound: linalool and limonene.
Solvent: DMSO
Dissolve the compounds with DMSO to set a series of concentration gradient of attractant.
Protocol:
Synchronize the nematodes.
Divide the NGM solid medium (d = 6 cm) equally into two parts (drawing on the surface of the culture dishes).
Put 50 \(\mu\)L compound of different concentration and DMSO as contrast respectively on the two parts. Make 3 repeats of each concentration.
Flush nematode from the plate with M9 and inoculate 20 \(\mu\)L (about 30 nematodes) into the center of the medium.
Incubate at 20°C for 1hr, and then place them at 4°C refrigerator for 1hr.
Count the nematodes of each part under the stereoscope.
Compare the results of different compound concentration and make a conclusion.
Detection of Killing Nematodes
Inoculate 5mL LB liquid mediums added Chl with experimental group E.coli BW25113 (transformed with rMpl or bace16 genes) and blank control group separately. Incubate at 37°C, 190 r.m.p for 3 hr.
Mix up 800\(\mu\)L control group and 200ul experimental liquid medium Inoculate 1mL mixture mentioned above to NGM plate. Incubate upside down at 37°C overnight.
Flush nematodes from the plate with M9, centrifugate (1500 r.p.m, 3min), abandon the supernatant and resuspend the precipitation with 3mL M9.
Add 500\(\mu\)L resuspended solution to the NGM plate inoculated with mixed E.coli as mentioned above as experimental group and NGM plate with blank E.coli as control group. 37°C incubate overnight.
Observe and compare the activation and size of nematodes of each group and draw a conclusion.
Nematode
Materials
NGM medium:
| Minimum Medium | ||||
|---|---|---|---|---|
| Reagent | NaCl | Bacto peptone（BD 4059002) | Agar | ddH2O |
| Dosage | 3 g | 2.5 g/L | 17 \(\mu\)L | 975 mL |
Prepare 1 mol/L CaCl2, 1 mol/L MgSO4 and phosphoric acid buffer meanwhile.
| Phosphoric Acid Buffer | |||
|---|---|---|---|
| Reagent | KH2PO4 | KHPO4 | ddH2O |
| Dosage | 108.3 g | 35.6 g/L | 1 L |
Autoclave the above mentioned reagents with 120°C for 30 min and then water bath them to 65°C.
Dissolve the cholesterol with ethanol to 5 mg/mL.
Add 1ml cholesterol, CaCl2, MgSO4 and 25 mL phosphoric acid buffer into the minimum medium in order (all at 65°C, shake up).
Make plate with the NGM medium, storing at 4°C.
OP50 E.coli deficit type (food of nematodes):
Streak inoculate OP50 on LB solid medium, Incubate at 37°C for 6 hr or overnight. Inoculate LB liquid medium with OP50 single colonies, and incubate (37°C, 220 r.p.m) overnight.
Cultivate
The incubation condition of caenorhabditis elegans N2 wild type:
Generally incubated at 20°C. Grow slowly at 16°C and grow fast at 25°C while egg laying amount declines.
Protocol:
Inoculate NGM medium with 150~200 \(\mu\)L OP50, incubate at 37°C for 12hr. Cut down a square of NGM(about 1cm x 1cm) with nematodes.
Put the square on the NGM medium with OP50 and let the surface with nematodes adown to contact the medium.
Seal and incubate upside down at 20°C.
Watch the growth condition of nematodes under the stereoscope everyday and re-inoculate every 4 to 5 days to avoid the nematodes growing too densely.
For inoculating abundant nematodes rapidly, or changing plate to provide more food and better condition for them, we can flush them with M9 and centrifuge them with 1500 r.m.p for 1 min, and incubate the precipitate on a new plate.
Synchronization
Materials:
| M9 NS | |||||
|---|---|---|---|---|---|
| Reagent | Na2HPO4 | KH2PO4 | NaCl | 1M MgSO4 | ddH2O |
| Dosage | 6 g | 3 g | 5 g | 1 mL | 1 L |
| Bleach Buffer | |||
|---|---|---|---|
| Reagent | NaOCl | 5M NaOH | ddH2O |
| Dosage | 50 \(\mu\)L | 100 \(\mu\)L | 850 \(\mu\)L |
Protocols:
We have tried several methods and two of them succeed as mentioned below, we recommend the first one by comparison.
1. Flush and incubate
Flush the nematodes with M9 NS
Centrifugation (3000 r.m.p, 1min)
Abandon the supernatant, add 1 mL bleach buffer to the precipitation
Centrifugation (3000 r.m.p, 1min), abandon the supernatant
Transfer the precipitation (nematode eggs) to a NGM plate with OP50, then the eggs will incubate synchronously.
2. Directly pick
Transfer 30 nematodes that are under egg laying period to a new NGM plate with OP50. Generally, the egg laying nematodes can lay about 8 eggs per hour. Remove all nematodes after 2hr.
Incubate the plate at 20°C for 3d and therefor can get about 300 to 400 nematodes.
Note: all of the procedure outlined above must be conducted under sterile condition.
Isolate nematodes from soil
modified Baermann funnel method:
1. Place a glass funnel (d = 10~15 cm) on the funnel stand linked with about 10cm rubber tube which is furnished with a flatjaw pinchcock.
2. Cut the soil sample into matchstick shape. Package 10 g(wet weight) with gauze and put it into the funnel, and then add water to immersion the sample.
3. After 4~24 hr, nematodes leave the plant tissue and swim in the water and ultimately precipitate at the bottom due to their gravity and hydrotaxis.
4. Open the flatjaw pinchcock, adopt about 5~15 mL liquid sample from the bottom which obtains most active nematodes of the soil sample. Check under the stereoscope and count nematodes, or centrifugate(1500 r) first and check the precipitation if the nematodes are too few.