Difference between revisions of "Part:BBa K4712104"

 
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<partinfo>BBa_K4712104 parameters</partinfo>
 
<partinfo>BBa_K4712104 parameters</partinfo>
 
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 +
 +
Protocol:
 +
Apparatus:
 +
Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip
 +
Materials:
 +
1. DNA Isothermal Amplification Reagent Kit (EasyGene Biotechnology)
 +
2. ddH2O
 +
3. Isothermal Amplification Specific Primers:
 +
 +
<table>
 +
<thead>
 +
  <tr>
 +
    <th>H5N1-F1</th>
 +
    <th>H5N1-R1</th>
 +
    <th>H5N1-F2</th>
 +
    <th>H5N1-R2</th>
 +
    <th>H5N1-F3</th>
 +
    <th>H5N1-R3</th>
 +
    <th>H5N1-F4</th>
 +
    <th>H5N1-R4</th>
 +
  </tr>
 +
</thead>
 +
<tbody>
 +
  <tr>
 +
    <td>H5N6-F1</td>
 +
    <td>H5N6-R1</td>
 +
    <td>H5N6-F2</td>
 +
    <td>H5N6-R2</td>
 +
    <td>H5N6-F3</td>
 +
    <td>H5N6-R3</td>
 +
    <td>H5N6-F4</td>
 +
    <td>H5N6-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Mtu-F1</td>
 +
    <td>Mtu-R1</td>
 +
    <td>Mtu-F2</td>
 +
    <td>Mtu-R2</td>
 +
    <td>Mtu-F3</td>
 +
    <td>Mtu-R3</td>
 +
    <td>Mtu-F4</td>
 +
    <td>Mtu-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>NME-F1</td>
 +
    <td>NME-R1</td>
 +
    <td>NME-F2</td>
 +
    <td>NME-R2</td>
 +
    <td>NME-F3</td>
 +
    <td>NME-R3</td>
 +
    <td>NME-F4</td>
 +
    <td>NME-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SARS-CoV-F1</td>
 +
    <td>SARS-CoV-R1</td>
 +
    <td>SARS-CoV-F2</td>
 +
    <td>SARS-CoV-R2</td>
 +
    <td>SARS-CoV-F3</td>
 +
    <td>SARS-CoV-R3</td>
 +
    <td>SARS-CoV-F4</td>
 +
    <td>SARS-CoV-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>SARS-CoV-F5</td>
 +
    <td>SARS-CoV-R5</td>
 +
    <td>SARS-CoV-F6</td>
 +
    <td>SARS-CoV-R6</td>
 +
    <td>SARS-CoV-F7</td>
 +
    <td>SARS-CoV-R7</td>
 +
    <td>SARS-CoV-F8</td>
 +
    <td>SARS-CoV-R8</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Cd-F1</td>
 +
    <td>Cd-R1</td>
 +
    <td>Cd-F2</td>
 +
    <td>Cd-R2</td>
 +
    <td>Cd-F3</td>
 +
    <td>Cd-R3</td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
  <tr>
 +
    <td>SP-F1</td>
 +
    <td>SP-R1</td>
 +
    <td>SP-F2</td>
 +
    <td>SP-R2</td>
 +
    <td>SP-F3</td>
 +
    <td>SP-R3</td>
 +
    <td>SP-F4</td>
 +
    <td>SP-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>HAdV-B-F1</td>
 +
    <td>HAdV-B-R1</td>
 +
    <td>HAdV-B-F2</td>
 +
    <td>HAdV-B-R2</td>
 +
    <td>HAdV-B-F3</td>
 +
    <td>HAdV-B-R3</td>
 +
    <td>HAdV-B-F4</td>
 +
    <td>HAdV-B-R4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>HAdV-C-F1</td>
 +
    <td>HAdV-C-R1</td>
 +
    <td>HAdV-C-F2</td>
 +
    <td>HAdV-C-R2</td>
 +
    <td>HAdV-C-F3</td>
 +
    <td>HAdV-C-R3</td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
4. DL500 marker
 +
5. Test Sample: DNA Template（pUC57-M1）Concentration：1000cps/μL
 +
Storage: -20℃
 +
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>ddH2O</td>
 +
    <td></td>
 +
    <td>To 18</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Starter (10X)</td>
 +
    <td></td>
 +
    <td>2</td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
2. Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing).
 +
3. Incubate at 37°C for 20 minutes.
 +
4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis.
 +
Results:
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig8f.png
 +
 +
The efficiency verification of crRNAs targeting the DNA sequences of HAdV-B. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig9f.png
 +
 +
Fluorescence intensity of crRNAs targeting HAdV-B after CRISPR reaction under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig10g-left.png
 +
 +
https://static.igem.wiki/teams/4712/wiki/result/fig10g-right.png
 +
 +
Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting HAdV-B after one-tube reaction of RPA and CRISPR under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L

Revision as of 13:12, 12 October 2023


HAdV-B-F3

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 Human adenovirus B1 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 Reagent Kit (EasyGene Biotechnology) 2. ddH2O 3. Isothermal Amplification Specific Primers:

<thead> </thead> <tbody> </tbody>
H5N1-F1 H5N1-R1 H5N1-F2 H5N1-R2 H5N1-F3 H5N1-R3 H5N1-F4 H5N1-R4
H5N6-F1 H5N6-R1 H5N6-F2 H5N6-R2 H5N6-F3 H5N6-R3 H5N6-F4 H5N6-R4
Mtu-F1 Mtu-R1 Mtu-F2 Mtu-R2 Mtu-F3 Mtu-R3 Mtu-F4 Mtu-R4
NME-F1 NME-R1 NME-F2 NME-R2 NME-F3 NME-R3 NME-F4 NME-R4
SARS-CoV-F1 SARS-CoV-R1 SARS-CoV-F2 SARS-CoV-R2 SARS-CoV-F3 SARS-CoV-R3 SARS-CoV-F4 SARS-CoV-R4
SARS-CoV-F5 SARS-CoV-R5 SARS-CoV-F6 SARS-CoV-R6 SARS-CoV-F7 SARS-CoV-R7 SARS-CoV-F8 SARS-CoV-R8
Cd-F1 Cd-R1 Cd-F2 Cd-R2 Cd-F3 Cd-R3
SP-F1 SP-R1 SP-F2 SP-R2 SP-F3 SP-R3 SP-F4 SP-R4
HAdV-B-F1 HAdV-B-R1 HAdV-B-F2 HAdV-B-R2 HAdV-B-F3 HAdV-B-R3 HAdV-B-F4 HAdV-B-R4
HAdV-C-F1 HAdV-C-R1 HAdV-C-F2 HAdV-C-R2 HAdV-C-F3 HAdV-C-R3

4. DL500 marker 5. Test Sample: DNA Template（pUC57-M1）Concentration：1000cps/μL Storage: -20℃ 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
ddH2O To 18
Starter (10X) 2

2. Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing). 3. Incubate at 37°C for 20 minutes. 4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis. Results:

fig8f.png

The efficiency verification of crRNAs targeting the DNA sequences of HAdV-B. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

fig9f.png

Fluorescence intensity of crRNAs targeting HAdV-B after CRISPR reaction under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

fig10g-left.png

fig10g-right.png

Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting HAdV-B after one-tube reaction of RPA and CRISPR under Bright and UV illumination. The figures utilize pseudocolor to facilitate analysis using software (Image Lab 6). No crRNA is added into negative control. The concentration of DNA template is 10nM/L