Difference between revisions of "Part:BBa K4712069"

 
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<partinfo>BBa_K4712069 parameters</partinfo>
 
<partinfo>BBa_K4712069 parameters</partinfo>
 
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Protocol:
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1. For a 20μL reaction system:
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<table><tr><th>Reagent</th><th>Stock Concentration</th><th>Volume Added（μL）</th></tr><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></table>
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2. Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing).
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3. Incubate at 37°C for 20 minutes.
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4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis.
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https://static.igem.wiki/teams/4712/wiki/result/fig7e.png
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Electrophoresis of RPA Primer Screening (1) for Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 (SARS-Cov)
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https://static.igem.wiki/teams/4712/wiki/result/fig9c.png
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The figure above correspond to the fluorescence intensity of crRNA targeting Severe acute respiratory Syndrome coronavirus 2 isolate Wuhan-Hu-1 after CRISPR reaction under Bright and UV illumination. The figure 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.
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https://static.igem.wiki/teams/4712/wiki/result/fig8c.png
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The linear graphs sequentially correspond to the efficiency verification of crRNAs targeting the DNA sequences of  SARS-CoV pathogen. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
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https://static.igem.wiki/teams/4712/wiki/result/fig10c-left.png
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https://static.igem.wiki/teams/4712/wiki/result/fig10c-right.png
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Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting SARS-CoV 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 12:38, 12 October 2023


SARS-CoV-R3

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 Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 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: 1. For a 20μL reaction system:

ReagentStock ConcentrationVolume Added（μL）
Forward Primer10μM1
Reverse Primer10μM1
Rehydration Buffer (2X)10
DNA Template10nM/L2
ddH2OTo 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.

fig7e.png

Electrophoresis of RPA Primer Screening (1) for Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 (SARS-Cov)

fig9c.png

The figure above correspond to the fluorescence intensity of crRNA targeting Severe acute respiratory Syndrome coronavirus 2 isolate Wuhan-Hu-1 after CRISPR reaction under Bright and UV illumination. The figure 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.

fig8c.png

The linear graphs sequentially correspond to the efficiency verification of crRNAs targeting the DNA sequences of SARS-CoV pathogen. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.

fig10c-left.png fig10c-right.png

Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting SARS-CoV 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.