RSV standard and copyback genomes PCR Protocols

*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 500ng of RNA diluted in dH₂O (range 200-2000ng (background increases with more RNA))

    `10µL` reaction: dilute in `4µL` dH<sub>2</sub>O 
   
   
   
    `20µL` reaction: dilute in `8µL` dH<sub>2</sub>O 
   

2. Add primer (2micromolar (µM))

    `10µL` reaction: `0.5µL` 
   
   
   
    `20µL` reaction: `1µL` 
   

3. Add dNTPs

    `10µL` reaction: `0.5µL` 
   
   
   
    `20µL` reaction: `1µL` 
   

4. Incubate at 65°C for 0h 10m 0s

    Program in PCR Machine: RT-DI1 
   

5. Prepare the mix

6. Add mix to sample

    `10µL` reaction: `5µL` 
   
   
   
    `20µL`reaction: `10µL` 
   

7. Incubate at 50°C for 0h 50m 0s then 85°C for 0h 5m 0s

    Program in PCR Machine: RT-DI2 
   

8. After program is finished keep at -20°C for at least 0h 20m 0s

9. Spin down tubes in mini microcentrifuge

10. Add RNase H (add in the PCR hood! DVGs are very stable!)

    `10µL` reaction: `0.5µL` 
    
    
    
    `20µL` reaction: `1µL` 
    

11. Incubate at 37°C for 0h 20m 0s

    Program in PCR Machine: RNaseH 
    

12. Keep in -20°C for at least 0h 20m 0s before moving on to the PCR

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 24µL of master mix to PCR tubes (can be done on bench)

3. Thaw cDNA samples and spin down in mini microcentrifuge

4. Add 1µLof cDNA sample to master mix (add in PCR hood! DVGs are very stable!)

5. Run PCR

Program in PCR Macine: RSVDI233

1. Prepare 1% agarose gel

    1 g pure agarose in `100mL` of 1X TAE buffer 
   
   
   
    Microwave until agarose solution dissolves completely (`0h 2m 0s`) 
   
   
   
    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 `1-5µL` 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 0h 30m 0s)

4. Place gel in electrophoresis chamber containing 1X TAE buffer (make sure buffer covers the gel)

5. Load ladder and samples to wells

    `6µL` of Ladder 
   
   
   
            Ladder stock recipe: 
   
              `100µL` GeneRuler 100 bp Plus DNA ladder from Thermo Scientific: SM0321 
   
   
   
              `100µL` DNA Gel Loading Dye (6X) from Thermo Scientific: R0611 
   
   
   
              `400µL` dH<sub>2</sub>O 
   
   
   
    `30µL` of Sample + Loading dye mix (dilutes from 6X to 1X) 
   
   
   
            `5µL` of DNA Gel Loading Dye (6X) from Thermo Scientific: R0611 
   
   
   
            `25µL` of sample 
   

6. Run at 110 volts for 0h 30m 0s

7. Analyze gel bands