TITLE: RSV standard and copyback genomes PCR Protocols
AUTHORS: Carolina Lopez

[DESCRIPTION]
Protocol for the detection of the Respiratory Syncytial Virus (RSV) standard and copyback genomes by PCR

[BEFORE_START]
PRINCIPLES BEHIND THE PROCEDURE MUST BE UNDERSTOOD. PLEASE CONSULT WITH EXPERIENCED LAB MEMBER THE FIRST TIME YOU USE THIS PROCEDURE. UPDATE AS A GENERAL PROCEDURE AS NECESSARY BUT DO NOT MODIFY WITH SPECIFICS TO YOUR PROJECT, INSTEAD DOWNLOAD AND PASTE A MODIFIED COPY IN YOUR NOTEBOOK.

[STEPS]
SECTION: RSV standard and copyback genomes PCR Protocols
1. *The “Big” DVG design can detect cbVGs with break before 14882 and rejoin before 15112 (RSVA2). The “Small” DVG design can detect cbVGs with break before 15112 and rejoin before 15194 (RSVA2). The “Small” DVG design should theoretically also pick up big DVGs, but bigger amplicons do not get amplified as well. “gRSV rev” has been tested as an RT primer for both “Big” and “Small” DVG design, however, gave an unspecific amplicon with the “Big” DVG design at around 500bp. As a consequence, two separate RT and PCR reactions must be run when wanting to look for both big DVGs and small DVGs. For most experiments, it is enough to only use the “Big” DVG design. RSV Genome can also be detected using this protocol when using the Genome primers. Primer locations are 387-410 and 894-918 (RSVA2), however not an optimal match (seems to have been designed for RSV B?). 

Procedure 
Reverse Transcription (RT): *RT reaction can be completed in a 10 μL reaction or a 20 μL reaction. The 10 μL reaction is preferred, if RNA concentrations allow, to help conserve reagents.

1. Start with 500 ng of RNA diluted in dH2O (range 200-2000ng (background increases with more RNA)) 
        10 µL reaction: dilute in 4 µL dH2O 
        20 µL reaction: dilute in 8 µL dH2O 
2. Add primer (2 micromolar (µM)) 
        10 µL reaction: 0.5 µL 
        20 µL reaction: 1 µL 
3. Add dNTPs 
        10 µL reaction: 0.5 µL 
        20 µL reaction: 1 µL 
4. Incubate at 65 °C for 10 min 
        Program in PCR Machine: RT-DI1 
5. Prepare the mix
 
6. Add mix to sample 
        10 µL reaction: 5 µL 
        20 µLreaction: 10 µL 
7. Incubate at 50 °C for 50 min then 85 °C for 5 min 
        Program in PCR Machine: RT-DI2 
8. After program is finished keep at -20 °C for at least 20 min 
9. Spin down tubes in mini microcentrifuge 
10. Add RNase H (add in the PCR hood! DVGs are very stable!) 
        10 µL reaction: 0.5 µL 
        20 µL reaction: 1 µL 
11. Incubate at 37 °C for 20 min 
        Program in PCR Machine: RNaseH 
12. Keep in -20 °C for at least 20 min before moving on to the PCR
SECTION: PCR
2. 1. Prepare master mix (can be done on bench) 
*Volumes below are for when 1 μL of cDNA is used. Adjust dH2O volume accordingly if more cDNA is added. Up to 4 μL cDNA can be added but increases background. Total reaction volume should equal 25 μL. 
 
2. Add 24 µL of master mix to PCR tubes (can be done on bench)
3. Thaw cDNA samples and spin down in mini microcentrifuge 
4. Add 1 µLof cDNA sample to master mix (add in PCR hood! DVGs are very stable!) 
5. Run PCR 

Program in PCR Macine: RSVDI233
 

SECTION: Gel Electrophoresis:
3. 1. Prepare 1% agarose gel 
        1 g pure agarose in 100 mL of 1X TAE buffer 
        Microwave until agarose solution dissolves completely (2 min) 
        Let agarose solution cool before adding Ethidium bromide (if you can keep your fingers on the flask without burning, then it is at an appropriate temperature) 
        Add 1-5 µL of Ethidium bromide and mix by swirling flask 
2. Pour agarose solution into gel cast (remember to put in the well comb) 
3. Let the agarose solidify (wait at least 30 min) 
4. Place gel in electrophoresis chamber containing 1X TAE buffer (make sure buffer covers the gel)
5. Load ladder and samples to wells 
        6 µL of Ladder 
                Ladder stock recipe: 
                  100 µL GeneRuler 100 bp Plus DNA ladder from Thermo Scientific: SM0321 
                  100 µL DNA Gel Loading Dye (6X) from Thermo Scientific: R0611 
                  400 µL dH2O 
        30 µL of Sample + Loading dye mix (dilutes from 6X to 1X) 
                5 µL of DNA Gel Loading Dye (6X) from Thermo Scientific: R0611 
                25 µL of sample 
6. Run at 110 volts for 30 min 
7. Analyze gel bands