Team:OUC-China/Notebook Protocols

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Molecular Tests

I Electroporation

A. Prepare appropriative competent cells E.coil BL21 for electroporation.

1.Inoculate E.coli BL21 with 100ml fresh Luria-Bertani medium Shaking culture to OD600 ： 0.5 to 1.0.

2.Put the mixture into 2 of 50ml centrifugal tube and store on ice for 15 min.

3.Centrifugation at 5000×g, 4℃ for 15min, discard the supernatant.

4.Resuspend the mixture by 33ml ultrapure water (ice-bath). Centrifuge at 5000×g, 4℃ for 15min, discard the supernatant.

5.Repeat step 4 for 2 times.

6.Resuspend the mixture by 20ml 10% sterile glycerol (ice-bath). centrifuge at 5000×g, 4℃ for 15min, discard the supernatant.

7.Resuspend the mixture with 1ml 10% sterile glycerol (ice-bath).

8.Subpackage the competent cells (100μl) into EP tube and store them at -80℃.

B. Electroporation

1.Put the competent cells E.coil BL21 on ice for 15min. put plasmid RP4 and interfere DNA on ice before use.

2.Mix 5μl RP4 and 5μl interference DNA with each tube of competent cells. And suck up them into cuvette (ice-bath).

3.Electroporation:5ms impulse wave (200Ω, 25μ Fd, the voltage was 2.1 kV)

4.Add 1ml SOC medium into the cuvette.

5.Culture the mixture with Luria-Bertani medium, 37℃ for 1h.

6.Plate the mixture on LB solid medium, 34μg/ml Chloramphenicol added. Static culture at 37℃ for 12h~14h.

II Dnases degradation

1.Incubate at 37℃ for 1h, after mixing the protein and plasmid(1μg). The mass ratio between protein and plasmid is 0, 2, 4, 6, 8, 10.

2.Put 1μl DNaseI (TaKaLa) and 10Xbuffer in every EP tube for 30mins.

3.1% agarose gel electrophoresis to test DNA degradation.

III Total RNA extraction reagent

1.Get zebrafish's tissue.

2.Homogenize by adding appropriate amount of RNAiso Plus (1mg sample using 1.25ml RNAiso Plus).

3.Keep the homogenate at room temperature for 5minutes.

4.Centrifuge at 12000Xg for 5 minutes at 4℃.

5.Transfer the supernatant to new centrifuge tube.

6.Add chloroform of 0.2 volume of RNAiso Plus used.

7.Vortex vigorously.

8.Keep at room temperature for 5minutes.

9.Centrifuge at 12000Xg for 15 minutes at 4℃.

10.Transfer the upper layer to a new centrifuge tube.

11.Add isopropanol of 0.5-1.0 volume of RNAiso Plus used.

12.Keep at room temperature for 10 minutes.

13.Centrifuge at 12000Xg for 10 minutes at 4℃.

14.Wash the RNA with equivalent amount of 75% ethanol.

15.Centrifuge at 7500Xg for 5 minutes at 4℃.

16.Discard supernatant and keep precipitate.

17.Air dry, do not heat to dry precipitate.

18.Dissolve with appropriate amount of DEPC-treated water.

IV RT-PCR

1.Prepare the following reaction mixture

dNTP Mixture (10mM each) 1μl

Oligo dT Primer (2.5uM) 1μl

Template RNA 8μl

2.Place tubes in the Thermal Cycler and set the parameters according to the following conditions for denaturation and annealing.

65℃ for 5min.

4℃

3.Prepare the reagent mixture for use in step 2 by combining the reagents in the proportions shown as below.

Reaction mixture used for denaturation and annealing from A-2 10μl

5X PrimeScript Buffer 4μl

RNase Inhibitor (40U/μl) 0.5μl

PrimeScript RTase (for 2 step) 0.5μl

RNase Free dH2O 5μl

4.Place the tubes in a Thermal Cycler and perform reverse transcription using the following program

42℃ 15-30min

95℃ 5min

4℃

5.Prepare reaction miture by combining the following reagents.

10XPCR Buffer II 5μl

dNTP Mixture(10mM each) 2μl

Upstream Primer 0.5μl

Downstream Primer 0.5μl

TaKaRa Ex Taq HS 0.5μl

Reverse transcriptant from step4 5μl

H20 up to 50μl

6.After mixing reagents, put all tubes in the thermal cycler, and start PCR under the following program.

94℃ 30s

57℃ 30s

72℃ 30s

For 30 cycles

7.After PCR, load 2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.

V Test of lysis device

1.Grow the culture at 37℃ with LB media for 12h.

2.Dilute it into one fourth and then split into several aliquots. A range of concentrations of arabinose was added.

3.Incubate at 37℃ again.

4.Set a time gradient and measure the absorbance at 600nm.

Cellular Test

Conjugation

1.Inoculate E.coli HB101（RP4,OriTRP4-RFP）in Luria-Bertani medium, 10μg/ml kanamycin added.

Shake cultivation at 37 ℃ for 8h to exponential phase.

2.Inoculate Vibrio harveyi (no plasmid) in 2216E.

Shake cultivation at 28 ℃ for 8h to exponential phase.

3.Mixed them (donor/recipient=1:3) in an EP tube, Static culture at 28℃ for 3h.

4.Plate the mixture and Vibrio harveyi respectively on TCBS agar medium, 10μg/ml kanamycin added. Static culture at 28℃ for 12h~14h.

5.Assay positive clone with plasmid extraction.

Comments:

1.The conjugation experiment consists of four parts:

a. E.coli HB101 (RP4, OriTRP4-RFP) and E.coli Top10.

b. E.coli HB101 (RP4, OriTR-RFP) and E.coli Top10.

c. E.coli HB101 (RP4, OriTRP4-RFP) and Vibrio harveyi.

d. E.coli HB101 (RP4, OriTR-RFP) and Vibrio harveyi.

2.The conjugation of the other parts is similar to the protocol as described previously.

3.Use appropriate medium, add appropriate antibiotics and select the appropriate culture environment according to specific experiment.

• Molecular Tests
• Cellular Test