Primer

Part:BBa_K4712032

Designed by: Ke Zhang   Group: iGEM23_SMS-Shenzhen   (2023-09-28)
Revision as of 12:20, 12 October 2023 by AvA0607 (Talk | contribs)


H5N1-F1

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 A virus(2004H5N1) 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.Apparatus: Thermal Cycler, Centrifuge, Fluorescence Quantitative PCR Instrument (Ya Rui), 2mL reaction tube, Pipette and Pipette Tip Result

fig7a.png

Electrophoresis of RPA Primer Screening for Influenza A H5N1 gene

fig9a.png

fig10a-left.png

fig10a-right.png

Figure 10. Linear graphs and figures (a) correspond to the fluorescence intensity of crRNAs targeting H5N1 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.

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