Difference between revisions of "Part:BBa K4712026"

 
Line 17: Line 17:
 
<partinfo>BBa_K4712026 parameters</partinfo>
 
<partinfo>BBa_K4712026 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
Protocol
 +
Apparatus:
 +
Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip
 +
Materials:
 +
1. DNA Isothermal Amplification Kit (EZ-Life Biotechnology)
 +
2. ddH2O
 +
3. Thermostatic amplification specific primers:
 +
  INFB-F1、 INFB-R1
 +
  INFB-F2、 INFB-R2
 +
  INFB-F3、 INFB-R3
 +
  INFB-F4、 INFB-R4
 +
  INFB-F5、 INFB-R5
 +
  H1N1-F6、H1N1-R6
 +
  H1N1-F7、H1N1-R7
 +
  H1N1-F8、H1N1-R8
 +
4. DL500 marker
 +
5. Test sample: DNA Template（pUC57-M1）Concentration：1000cps/μL
 +
Storage: -20°C
 +
Methods:
 +
1. For a 20μL reaction system:
 +
 +
<table>
 +
<thead>
 +
  <tr>
 +
    <th>Reagent</th>
 +
    <th>Stock Concentration</th>
 +
    <th>Volume Added（μL）</th>
 +
  </tr>
 +
</thead>
 +
<tbody>
 +
  <tr>
 +
    <td>Forward Primer</td>
 +
    <td>10μM</td>
 +
    <td>1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Reverse Primer</td>
 +
    <td>10μM</td>
 +
    <td>1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Rehydration Buffer (2X)</td>
 +
    <td></td>
 +
    <td>10</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DNA Template</td>
 +
    <td>10nM/L</td>
 +
    <td>2</td>
 +
  </tr>
 +
  <tr>
 +
    <td>H2O</td>
 +
    <td></td>
 +
    <td>To 18</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Starter (10X)</td>
 +
    <td></td>
 +
    <td>2</td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
2. Gently tap multiple times to mix the ingredient, and then centrifuge briefly (mix gently, avoiding vigorous shaking or vortexing).
 +
3. Incubate at 37 ℃ for 20 minutes.
 +
4. After heating at 65 ℃ for 10 minutes, the adhesive will run off.
 +
 +
Result:
 +
 +
<table>
 +
<thead>
 +
  <tr>
 +
    <th>Electrophoresis swimlane</th>
 +
    <th>Content</th>
 +
  </tr>
 +
</thead>
 +
<tbody>
 +
  <tr>
 +
    <td>1</td>
 +
    <td>DL500 maker</td>
 +
  </tr>
 +
  <tr>
 +
    <td>2</td>
 +
    <td>INFB-F1、 INFB-R1</td>
 +
  </tr>
 +
  <tr>
 +
    <td>3</td>
 +
    <td>INFB-F2、 INFB-R2</td>
 +
  </tr>
 +
  <tr>
 +
    <td>4</td>
 +
    <td>INFB-F3、 INFB-R3</td>
 +
  </tr>
 +
  <tr>
 +
    <td>5</td>
 +
    <td>INFB-F4、 INFB-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>6</td>
 +
    <td>INFB-F6、 INFB-R6</td>
 +
  </tr>
 +
  <tr>
 +
    <td>7</td>
 +
    <td>INFB-F5、 INFB-R5</td>
 +
  </tr>
 +
  <tr>
 +
    <td>8</td>
 +
    <td>H1N1-F6、H1N1-R6</td>
 +
  </tr>
 +
  <tr>
 +
    <td>9</td>
 +
    <td>H1N1-F7、H1N1-R7</td>
 +
  </tr>
 +
  <tr>
 +
    <td>10</td>
 +
    <td>H1N1-F8、H1N1-R8</td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig1b.png
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig10h-left.png
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig10h-right.png
 +
 +
Figure 10. Linear graphs and figures (h) correspond to the fluorescence intensity of crRNAs targeting INFB after one-tube reaction of RPA and CRISPR under Bright and UV illumination. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

Revision as of 12:07, 12 October 2023


INFB-F6

The primers were designed using NCBI BLAST and SanpGene to achieve efficient and specific amplification. This primer in RPA serves as the initial binding point for the amplification process, ensuring the specificity of the reaction by targeting the desired Influenza B virus DNA or RNA sequences. The primers provided data for mathematical modeling for further primer design.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Protocol Apparatus: Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip Materials: 1. DNA Isothermal Amplification Kit (EZ-Life Biotechnology) 2. ddH2O 3. Thermostatic amplification specific primers: 

 INFB-F1、 INFB-R1
 INFB-F2、 INFB-R2
 INFB-F3、 INFB-R3
 INFB-F4、 INFB-R4
 INFB-F5、 INFB-R5
 H1N1-F6、H1N1-R6
 H1N1-F7、H1N1-R7
 H1N1-F8、H1N1-R8

4. DL500 marker 5. Test sample: DNA Template（pUC57-M1）Concentration：1000cps/μL Storage: -20°C Methods: 1. For a 20μL reaction system:

<thead> </thead> <tbody> </tbody>
Reagent Stock Concentration Volume Added（μL）
Forward Primer 10μM 1
Reverse Primer 10μM 1
Rehydration Buffer (2X) 10
DNA Template 10nM/L 2
H2O To 18
Starter (10X) 2

2. Gently tap multiple times to mix the ingredient, and then centrifuge briefly (mix gently, avoiding vigorous shaking or vortexing). 3. Incubate at 37 ℃ for 20 minutes. 4. After heating at 65 ℃ for 10 minutes, the adhesive will run off.

Result:

<thead> </thead> <tbody> </tbody>
Electrophoresis swimlane Content
1 DL500 maker
2 INFB-F1、 INFB-R1
3 INFB-F2、 INFB-R2
4 INFB-F3、 INFB-R3
5 INFB-F4、 INFB-R4
6 INFB-F6、 INFB-R6
7 INFB-F5、 INFB-R5
8 H1N1-F6、H1N1-R6
9 H1N1-F7、H1N1-R7
10 H1N1-F8、H1N1-R8

fig1b.png

fig10h-left.png

fig10h-right.png

Figure 10. Linear graphs and figures (h) correspond to the fluorescence intensity of crRNAs targeting INFB after one-tube reaction of RPA and CRISPR under Bright and UV illumination. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.