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Team:OUC-China/Notebook Protocols
From 2014.igem.org
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| - | < | + | <h2>Molecular Tests</h2> |
| - | < | + | <h3>I Electroporation</h3> |
| - | < | + | <h4>A. Prepare appropriative competent cells E.coil BL21 for electroporation.</h4> |
<p>1.Inoculate E.coli BL21 with 100ml fresh Luria-Bertani medium Shaking culture to OD600 ： 0.5 to 1.0.</p> | <p>1.Inoculate E.coli BL21 with 100ml fresh Luria-Bertani medium Shaking culture to OD600 ： 0.5 to 1.0.</p> | ||
<p>2.Put the mixture into 2 of 50ml centrifugal tube and store on ice for 15 min.</p> | <p>2.Put the mixture into 2 of 50ml centrifugal tube and store on ice for 15 min.</p> | ||
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<p>7.Resuspend the mixture with 1ml 10% sterile glycerol (ice-bath).</p> | <p>7.Resuspend the mixture with 1ml 10% sterile glycerol (ice-bath).</p> | ||
<p>8.Subpackage the competent cells (100μl) into EP tube and store them at -80℃.</p> | <p>8.Subpackage the competent cells (100μl) into EP tube and store them at -80℃.</p> | ||
| - | < | + | <h4>B. Electroporation</h4> |
<p>1.Put the competent cells E.coil BL21 on ice for 15min. put plasmid RP4 and interfere DNA on ice before use.</p> | <p>1.Put the competent cells E.coil BL21 on ice for 15min. put plasmid RP4 and interfere DNA on ice before use.</p> | ||
<p>2.Mix 5μl RP4 and 5μl interference DNA with each tube of competent cells. And suck up them into cuvette (ice-bath). </p> | <p>2.Mix 5μl RP4 and 5μl interference DNA with each tube of competent cells. And suck up them into cuvette (ice-bath). </p> | ||
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<p>5.Culture the mixture with Luria-Bertani medium, 37℃ for 1h.</p> | <p>5.Culture the mixture with Luria-Bertani medium, 37℃ for 1h.</p> | ||
<p>6.Plate the mixture on LB solid medium, 34μg/ml Chloramphenicol added. Static culture at 37℃ for 12h~14h.</p> | <p>6.Plate the mixture on LB solid medium, 34μg/ml Chloramphenicol added. Static culture at 37℃ for 12h~14h.</p> | ||
| + | <h3>II Dnases degradation</h3> | ||
| + | <p>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.</p> | ||
| + | <p>2.Put 1μl DNaseI (TaKaLa) and 10Xbuffer in every EP tube for 30mins.</p> | ||
| + | <p>3.1% agarose gel electrophoresis to test DNA degradation.</p> | ||
| + | <h3>III Total RNA extraction reagent</h3> | ||
| + | <p>1.Get zebrafish's tissue.</p> | ||
| + | <p>2.Homogenize by adding appropriate amount of RNAiso Plus (1mg sample using 1.25ml RNAiso Plus).</p> | ||
| + | <p>3.Keep the homogenate at room temperature for 5minutes.</p> | ||
| + | <p>4.Centrifuge at 12000Xg for 5 minutes at 4℃.</p> | ||
| + | <p>5.Transfer the supernatant to new centrifuge tube.</p> | ||
| + | <p>6.Add chloroform of 0.2 volume of RNAiso Plus used.</p> | ||
| + | <p>7.Vortex vigorously.</p> | ||
| + | <p>8.Keep at room temperature for 5minutes.</p> | ||
| + | <p>9.Centrifuge at 12000Xg for 15 minutes at 4℃.</p> | ||
| + | <p>10.Transfer the upper layer to a new centrifuge tube.</p> | ||
| + | <p>11.Add isopropanol of 0.5-1.0 volume of RNAiso Plus used.</p> | ||
| + | <p>12.Keep at room temperature for 10 minutes.</p> | ||
| + | <p>13.Centrifuge at 12000Xg for 10 minutes at 4℃.</p> | ||
| + | <p>14.Wash the RNA with equivalent amount of 75% ethanol.</p> | ||
| + | <p>15.Centrifuge at 7500Xg for 5 minutes at 4℃.</p> | ||
| + | <p>16.Discard supernatant and keep precipitate.</p> | ||
| + | <p>17.Air dry, do not heat to dry precipitate.</p> | ||
| + | <p>18.Dissolve with appropriate amount of DEPC-treated water.</p> | ||
| + | |||
Revision as of 18:16, 17 October 2014
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.
