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Team:OUC-China/Notebook Protocols
From 2014.igem.org
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| - | < | + | <h1 id="Molecular_Tests">Molecular Tests</h1> |
| - | < | + | <h2><p class="text-primary“>I Electroporation</p2></h2> |
<h4>A. Prepare appropriative competent cells <em>E.coil</em> BL21 for electroporation.</h4> | <h4>A. Prepare appropriative competent cells <em>E.coil</em> BL21 for electroporation.</h4> | ||
<p>1.Inoculate <em>E.coli</em> BL21 with 100ml fresh Luria-Bertani medium Shaking culture to OD600 ： 0.5 to 1.0.</p> | <p>1.Inoculate <em>E.coli</em> BL21 with 100ml fresh Luria-Bertani medium Shaking culture to OD600 ： 0.5 to 1.0.</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> | ||
| - | < | + | <h2><p class="text-primary“>II Dnases degradation</p></h2> |
<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>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>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> | <p>3.1% agarose gel electrophoresis to test DNA degradation.</p> | ||
| - | < | + | <h2><p class="text-primary“>III Total RNA extraction reagent</p></h2> |
<p>1.Get zebrafish's tissue.</p> | <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>2.Homogenize by adding appropriate amount of RNAiso Plus (1mg sample using 1.25ml RNAiso Plus).</p> | ||
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<p>17.Air dry, do not heat to dry precipitate.</p> | <p>17.Air dry, do not heat to dry precipitate.</p> | ||
<p>18.Dissolve with appropriate amount of DEPC-treated water.</p> | <p>18.Dissolve with appropriate amount of DEPC-treated water.</p> | ||
| - | < | + | <h2><p class="text-primary“>IV RT-PCR</p></h2> |
<p>1.Prepare the following reaction mixture</p> | <p>1.Prepare the following reaction mixture</p> | ||
<p> dNTP Mixture (10mM each) 1μl</p> | <p> dNTP Mixture (10mM each) 1μl</p> | ||
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<p> For 30 cycles</p> | <p> For 30 cycles</p> | ||
<p>7.After PCR, load 2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.</p> | <p>7.After PCR, load 2ul to a 1% agarose gel to have a quick run checking the production of desired fragment.</p> | ||
| - | < | + | <h2><p class="text-primary“>V Test of lysis device></p></h2> |
<p>1.Grow the culture at 37℃ with LB media for 12h. </p> | <p>1.Grow the culture at 37℃ with LB media for 12h. </p> | ||
<p>2.Dilute it into one fourth and then split into several aliquots. A range of concentrations of arabinose was added.</p> | <p>2.Dilute it into one fourth and then split into several aliquots. A range of concentrations of arabinose was added.</p> | ||
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<p>4.Set a time gradient and measure the absorbance at 600nm. </p> | <p>4.Set a time gradient and measure the absorbance at 600nm. </p> | ||
| - | < | + | <h1 id="Cellular_Test">Cellular Test</h1> |
| - | < | + | <h2><p class="text-primary“>Conjugation</p></h2> |
<p>1.Inoculate <em>E.coli</em> HB101（RP4,OriTRP4-RFP）in Luria-Bertani medium, 10μg/ml kanamycin added. </p> | <p>1.Inoculate <em>E.coli</em> HB101（RP4,OriTRP4-RFP）in Luria-Bertani medium, 10μg/ml kanamycin added. </p> | ||
<p> Shake cultivation at 37 ℃ for 8h to exponential phase. </p> | <p> Shake cultivation at 37 ℃ for 8h to exponential phase. </p> | ||
Revision as of 02:13, 18 October 2014
Molecular Tests
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.
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.
Cellular Test
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