Difference between revisions of "Part:BBa K4712062"
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<partinfo>BBa_K4712062 parameters</partinfo> | <partinfo>BBa_K4712062 parameters</partinfo> | ||
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| + | Protocol: | ||
| + | |||
| + | 1. For a 20μL reaction system: | ||
<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> | <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). | ||
| + | |||
| + | 3. Incubate at 37°C for 20 minutes. | ||
| + | |||
| + | 4. After heating at 65°C for 10 minutes, proceed to gel electrophoresis. | ||
https://static.igem.wiki/teams/4712/wiki/result/fig1c.png | https://static.igem.wiki/teams/4712/wiki/result/fig1c.png | ||
Revision as of 12:27, 12 October 2023
NME-F4
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 Neisseria meningitidis DNA or RNA sequences. The primers provided data for mathematical modeling for further primer design.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protocol:
1. For a 20μL reaction system:
| 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.
Electrophoresis of RPA Primer Screening for Neisseria meningitidis (NME)
The figure above correspond to the fluorescence intensity of crRNA targeting Neisseria meningitidis 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.
The linear graphs sequentially correspond to the efficiency verification of crRNAs targeting the DNA sequences of Neisseria meningitidis pathogen. No crRNA is added into negative control. The concentration of DNA template is 10nM/L.
Linear graphs and figure correspond to the fluorescence intensity of crRNAs targeting NME 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.
