Editing Recombinase Polymerase Amplification (section)

== Materials ==
<div id="section_body_materials"><br/>
{|
|-
| Name
| Company
| Catalog Number
| Comments
|-
| qRPA Assay
| <br/>
| <br/>
| <br/>
|-
| HIV-1 forward primer
| Integrated DNA Technologies
| custom DNA oligos
| 5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3’&nbsp;
|-
| HIV-1 reverse primer
| Integrated DNA Technologies
| custom DNA oligos
| 5’-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3’&nbsp;
|-
| HIV-1 probe
| BioSearch Technologies&nbsp;
| custom DNA oligos
| 5’- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C
[dT-BHQ1] GTA CCC CCC AAT CCC C -3’&nbsp;

|-
| IPC probe
| BioSearch Technologies&nbsp;
| custom DNA oligos
| 5’-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T
[dT-BHQ1] GGT TTT GTA ATT GGA A -3’

|-
| RPA exo reagents (pellets, rehydration buffer, magnesium
acetate

| TwistDx
| TwistAmp exo
| <br/>
|-
| PCR tube strips
| BioRad
| TLS0801
| <br/>
|-
| PCR flat cap tube strips
| BioRad
| TCS0803
| <br/>
|-
| Micro-seal adhesive
| BioRad
| 558/MJ&nbsp;
| <br/>
|-
| HIV-1 target (pHIV-IRES- eYFPΔEnvΔVifΔVpr)
| <br/>
| <br/>
| custom plasmid, see: Segall, H. I., Yoo, E. & Sutton, R.
E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118–129, doi:10.1016/S1525-0016(03)00134-5 (2003).

|-
| Human male genomic DNA
| Applied Biosystems
| 360486
| <br/>
|-
| 96 well cold-block
| Cole Parmer
| EW-36700-48
| <br/>
|-
| Thermal cycler
| BioRad
| CFX96
| <br/>
|-
| MiniFuge
| VWR
| 93000-196
| <br/>
|-
| Vortex
| VWR
| 58816-121
| <br/>
|-
| Tris buffer 1.0 M, pH 8.0
| EMD Millipore
| 648314
| <br/>
|-
| EDTA 0.5 M, pH 8.0
| Promega
| V4321
| <br/>
|-
| Nuclease free water
| Ambion
| AM9937
| <br/>
|-
| IPC Development
| <br/>
| <br/>
| <br/>
|-
| Cryptosporidium parvum IPC template
| Waterborne Inc
| P102C
| It is also possible to order a double stranded synthetic
target from IDT if the user is unequipped to work with C. parvum (a BSL-2 infectious agent). PCR and RPA primers for C. parvum were designed using GenBank accession number AF115377.1

|-
| PCR long forward primer
| Integrated DNA Technologies
| custom DNA oligos
| 5’-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA
AGG CAG CAG GC-3’

|-
| PCR long reverse primer
| Integrated DNA Technologies
| custom DNA oligos
| 5’- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG
AGT ATT CAA GGC ATA -3’

|-
| Phusion High-Fidelty DNA Polymerase
| New England Biolabs
| M0530S
| <br/>
|-
| Qiaquick Gel Extraction Kit
| Qiagen
| 28704
| <br/>
|-
| TAE 10X buffer
| EMD Millipore
| 574797
| <br/>
|-
| Agarose
| Sigma Aldrich
| A9539
| <br/>
|-
| Microscope Experiments
| <br/>
| <br/>
| <br/>
|-
| Upright fluorescence microscope
| Zeiss
| Zeiss Imager.J1
| <br/>
|-
| Stage heater
| Bioscience Tools
| TC-GSH
| <br/>
|-
| 1-Channel Precision High Stability Temperature Controller
| Bioscience Tools
| TC-1100S
| <br/>
|-
| FAM/GFP filter cube
| Zeiss
| filter set 38 (000000-1031-346)
| excitation BP 470/40 nm, emission BP 520/50 nm
|-
| HEX filter cube
| Chroma
| 49014
| excitation BP 530/30 nm, emission BP 575/40 nm
|-
| Laser cutter
| Engraver's Network
| VLS3.60
| <br/>
|-
| 1/8" black acrylic
| McMaster Carr
| 8505K113
| <br/>
|-
| 1.5 mm clear acrylic
| McMaster Carr
| PD-72268940&nbsp;
| <br/>
|-
| Super glue
| Office Depot
| Duro super glue&nbsp;
| <br/>
|-
| PCR grade mineral oil
| Sigma Aldrich
| M8662-5VL
| <br/>
|-
| Data Analysis
| <br/>
| <br/>
| <br/>
|-
| Microsoft Excel
| Microsoft
| <br/>
| <br/>
|-
| MATLAB
| MATLAB
| <br/>
| <br/>
|-
| MATLAB script: "JoVE_qRPA_standard_curve.m”<br/>
| Included in SI
| <br/>
| <br/>
|-
| MATLAB script: "JoVE_qRPA_validation_and_quantification.m”
| Included in SI
| <br/>
| <br/>
|-
| MATLAB script: "JoVE_real_time_intensity_to_excel.m”
| Included in SI
| <br/>
| <br/>
|-
| Adobe Illustrator
| Adobe
| <br/>
| <br/>
|-
| JoVE_qRPA_well.ai
| Included in SI
| <br/>
| <br/>
|-
| JoVE_qRPA_base.ai
| Included in SI
| <br/>
| <br/>
|-
| AxioVision software
| Zeiss
| <br/>
| <br/>
|-
| JoVE_AxioVision_Script.ziscript
| Included in SI
| <br/>
| <br/>
|}
<br/></div><div id="section_body_references">
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</div> <br/>&nbsp;[[Category:Protocols]]