Difference between revisions of "Part:BBa K4712063"

Revision as of 11:54, 12 October 2023

NME-R4

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

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:

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.

@@ Line 19: / Line 19: @@
 Protocol:
 . 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>
 . Gently tap to mix several times, briefly centrifuge, repeat 3 times (mix gently to avoid vigorous vortexing).
 . Incubate at 37°C for 20 minutes.
 . After heating at 65°C for 10 minutes, proceed to gel electrophoresis.
-Results:
-[Image]
-Electrophoresis of RPA Primer Screening for Neisseria meningitidis (NME)
-[Image]
-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.
-[Image]
-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.
-[Image]
-[Image]
-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.