Difference between revisions of "Team:HUST-China/wetlab/protocols"

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                     <li><a href="#section2">Reaction System<br> of Colony PCR</a></li>
 
                     <li><a href="#section2">Reaction System<br> of Colony PCR</a></li>
 
                     <li><a href="#section3">SDS-PAGE</a></li>
 
                     <li><a href="#section3">SDS-PAGE</a></li>
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                    <li><a href="#section3">Titration</a></li>
 
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                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3>
 
                     <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3>
 
                     <p><strong>The protocol of SDS-PAGE:</strong><p>
 
                     <p><strong>The protocol of SDS-PAGE:</strong><p>
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                     <p>a.Centrifuge 1ml culture medium/bacteria liquid at 12000rmp for 2 min to separate the supernatant and cells. </p>
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                     <p>1. Take 1ml of the bacterial suspension(OD600 is about 2.0), add 9ml LB medium,  culture the bacteria in a shaker  until its OD600 is 0.6. </p>
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                     <p>b.Add 100μl DNase/RNase-Free Water to the precipitation, then add in 100μl  2×loading buffer.</p>
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                    <p>2. Add 1 ‰ IPTG to induce our engineering bacteria, and culture them for 5 hours.</p>
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                     <p>c.Place tubes in 100℃ heat block for 10 min.</p>
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                     <p>3. Centrifuge the bacterial suspension, and gently rinse it, add utrapure water into the cultures until its OD600 is about 2.0.</p>
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                     <p>d.Centrifuge at 12000rmp for 2 min.</p>
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                     <p>4. Prepare different concentrations of terbium chloride solution of 40ml. Adding 3ml bacteria suspension to each of them respectively.</p>
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                     <p>e.Do SDS-PAGE</p>
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                     <p>5. Add excess sodium hydroxide to the supernatant until all the ions are precipitated. Centrifugate.</p>
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                     <p>6. The resulting precipitate was titrated with hydrogen chloride to give the titration results.</p>
 
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                <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> 
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                  <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Titration</h3>
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                <p>We transferred the plasmids carrying the sequences encoding 3×LBT into BL21, and set a control group in which 3×LBT is not involved in the plasmids transferred. Both can be expressed with the induction of IPTG. Having cultivated the bacteria into similar densities, we add IPTG and then incubate them for 5 hours. Afterwards, we boiled the bacteria to release the proteins on their cell membrane. Then we detected the proteins we extracted by SDS-PAGE. As a result, bacteria in the test group expressed a large quantity of LBT while those in the control group did not.
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Revision as of 18:26, 1 November 2017

Protocols

「Protocols」

Cloning methods

A.The PCR Reaction System

Components (50μL) Volume(μL)
5×phusion HF Buffer 10
dNTPs(2.5mM) 5
Primer-F(10μM) 2.5
Primer-R(10μM) 2.5
DMSO 1.5
phusion 0.5
ddH2O 27.5
Template 0.5

B.The double enzyme digestion system (Q.cut)

Components (30μl) Volume(μl)
10 x Q.cut buffer 3
Substrate 10
Enzyme I 1
Enzyme II 1
ddH2O 15
Conditions 37℃ 1.5~2h

C.Ligation system

Components (10μl) Volume(μl)
T4 ligase 1
T4 ligase buffer 1
Linearized Vector 1
Insert Gene 7
Conditions 16°C 1h

D.The SOE（Gene splicing by overlap extension） PCR

We use SOE to fuse different genes, which are difficult to cut or ligate. We achieve the fusing by overlap extension during PCR. First, we need to design 4 primers. Primer 1 & 4 are normal ones. The left part of primer 2 is normal primer, while the right part is primer for gene B. Primer 3 is just like primer 2. The right part of primer 3 is normal, while the left part is primer for gene A. Some base of primer 2 and primer 3 can reach complementary base pairing. Second, we amplify gene A by primer 1 & 2 and amplify gene B by primer 3 & 4 respectively. After that we can get extended gene A and gene B. Last but not least, we blend extended gene A & gene B can complemet each other and form the hybirdized strands. Under the function of DNA polymerase I, gene A and gene B can be the primer and templet of each other. After PCR, we can get fused Gene A-B.

E.Transformation into E. coli DH5α

The ligation product was transformed into E.coli DH5α strain.

If the vector was pET28a, the strain was grown in LB plate medium containing 10μg/ml kanamycin at 37°C.

If the vector was pBAD30, the strain was grown in LB plate medium containing 100μg/ml ampicillin at 37°C.

If the vector was pSB1C3, the strain was grown in LB plate medium containing 25μg/ml chloramphenicol at 37°C.

Reaction System of Colony PCR

Components (15μl) Volume(μl)
Es Taq Mix(2×) 7.5
Primer-F(10uM) 0.6
Primer-R(10uM) 0.6
ddH2O 6.3
Single Colony a little

Construction of the whole circuit

The protocol of SDS-PAGE:

1. Take 1ml of the bacterial suspension(OD600 is about 2.0), add 9ml LB medium, culture the bacteria in a shaker until its OD600 is 0.6.

2. Add 1 ‰ IPTG to induce our engineering bacteria, and culture them for 5 hours.

3. Centrifuge the bacterial suspension, and gently rinse it, add utrapure water into the cultures until its OD600 is about 2.0.

4. Prepare different concentrations of terbium chloride solution of 40ml. Adding 3ml bacteria suspension to each of them respectively.

5. Add excess sodium hydroxide to the supernatant until all the ions are precipitated. Centrifugate.

6. The resulting precipitate was titrated with hydrogen chloride to give the titration results.

Titration

We transferred the plasmids carrying the sequences encoding 3×LBT into BL21, and set a control group in which 3×LBT is not involved in the plasmids transferred. Both can be expressed with the induction of IPTG. Having cultivated the bacteria into similar densities, we add IPTG and then incubate them for 5 hours. Afterwards, we boiled the bacteria to release the proteins on their cell membrane. Then we detected the proteins we extracted by SDS-PAGE. As a result, bacteria in the test group expressed a large quantity of LBT while those in the control group did not.

Acknowledgments: