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

[DESCRIPTION]
Protocols for the detection of Sendai Virus (SeV) standard and copy-back 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: Reverse Transcription (RT)
1. *Different RT kits and steps for cbVG and Genome
    1/3) cbVG: Use SuperScript III Kit

Procedure
*RT reaction can be completed in a 10 µL reaction or a 20 µLreaction. The 10 µLreaction is preferred, if RNA concentrations allow, to help conserve reagents.
        1. Start with 1 µg of RNA 
                10 µL reaction: dilute in 4 µL dH2O 
                20 µL reaction: dilute in 8 µL dH2O 
        2. Pre-mix 10 micromolar (µM) primer and dNTPs  
                10 µL reaction: 0.5 µL of each, per sample 
                20 µL reaction: 1 µLof each, per sample
        3. Add Primer/dNTP mix to the RNA sample 
                10 µL reaction: 1 µL 
               20 µL reaction: 2 µL 
        4. Mix and spin down 
        5. Incubate at 65 °C for   10 min 
                Program in PCR Machine: RT-DI1 

        6. Prepare the mix (see TABLE below)

 

• Add the SIII enzyme separately to each sample, don’t include it in the mix.

        7. Add mix to sample, vortex briefly, and spin down 
                10 µL reaction: 5 µL 
                20 µL reaction: 10 µL 
        8. Incubate at 50 °C for 50 min then 85 °C for 5 min  
                Program in PCR Machine: RT-DI2 
        9. After program is finished keep at -20 °C or on ice for at least 5 min
        ————————————— Move to PCR Hood (for all later steps) 
        ————————————— (Clean PCR hood with 10% bleach. Wipe down pipette with bleach in between samples.)  
        10. Add RNase H 
                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 on ice for at least 5 min then you can do the PCR or store at -20 °C
SECTION: Reverse Transcription (RT)
2. *Different RT kits and steps for cbVG and Genome
        2/3) Genome: Use Roche Kit

Procedure
*RT reaction can be completed in a 10 µL reaction or a 20 µL reaction. The 10 ∝L  reaction is preferred, if RNA concentrations allow, to help conserve reagents.

        1. Start with 1000 ng of RNA diluted in dH2O  
                10 µL reaction: dilute in 3 µL dH2O 
                20 µL reaction: dilute in 6 µL dH2O 
        2. Add primer: 
                10 µL reaction: 3.5 µL  
                20 µL reaction: 7 µL 
        3. Incubate at 65 °C for 10 min  
                Program in PCR Machine: RT-DI1 
        4. Prepare the mix

 

        5. Add mix to sample
                10 µL reaction: 3.5 µL 
                20 µL reaction: 7 µL  
        6. Incubate at 50 °C for 60 minutes then 85 °C for 5 min then 4 °C forever 
                Program in PCR machine: RORT2SPE 
        7. Keep in -20 °C for at least 20 min before moving on to the PCR

SECTION: PCR
3. *Same kit and steps for both cbVG and Genome*
3/3) PCR

Procedure
        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 µL of cDNA sample to master mix (add in PCR hood! cbVG are very stable!)
        5. Run PCR 

Program in PCR Machine: DIPCR

 


SECTION: Gel Electrophoresis
4.         1. Prepare 1% agarose gel  
                1 g pure agarose in 100 mL of 1X TAE buffer 
                Microwave until agarose solution dissolves completely (~3 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 an electrophoresis chamber containing 1X TAE buffer (make sure buffer covers  gel) 
        5. Load ladder and samples to wells 
                6 µL of Ladder  
                               Ladder stock recipe:  
                              100 µL Gene Ruler 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 120 volts for 30-40 minutes 
        7. Analyze gel bands

SECTION: Sendai Virus Genomic and cbVG PCR CONCEPT
5.  


 


SeV copy back VG PCR strategy and validation. (A) Diagram of the genomic composition of the full-lengthSeV genome (gSeV) and of a representative copy-back VG of unknown length. Arrows indicate the location ofprimers used for RT and amplification (PCR). Full-length size of the genome is indicated. Expected amplicon  size of 760 nt of the gSeV to be detected through our PCR assay is indicated. This strategy allows detection of most copy-back VGs replicating in an infected cell. (B) Schematics of SeV strain Cantell's predominant 546 nt long cbVG. Expected amplicon size of 278 nt of this particular cbVG to be detected through our PCR assay isindicated. (C) Validation of the cbVG PCR assay. cbVG and gSeV amplicons from plasmids encoding the full-length SeV strain Cantell genome (lane 1) or the SeV strain Cantell dominant cbVG (lane 2) after amplificationusing the primers depicted in (A).https://doi.org/10.1371/journal.ppat.1003703.