Buffer recipes 6X SDS sample buffer(Laemmli buffer) Virology assays Plaque assay Molecular biology RNA extraction RT-PCR

-PCR Transfection siRNA transfection using Dharmafect Cell culture related Heat inactivate FBS Microscopy Immunofluorescent (22mm cover slip coated 35mm dish/6-well plate)

Plaque assay
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Seed VERO cells in 6-well plate Prepare 10-fold serial dilutions of virus stock (101 to 106) with serum-free medium Remove medium from the 6-well plate and wash twice with serum-free medium Add 200 l of 104,105,106-fold diluted virus to each well (duplicate or triplicate) Incubate the plate at 37oC for 1h, shake the plate every 5min (Go to 6 for 0.3% agarose overlay, Go to 17 for 1% CMC overlay)

Using 0.3% agarose overlay 6. 2 ml agarose overlay medium is needed for each well 7. Aliquot serum free medium to 50 ml falcon and warm in water bath at 37 C 8. Before the end of the 1h incubation, mix 3% agarose and serum free medium v:v=1:9 9. Keep the agarose overlay medium in 37 C water bath to prevent from solidifying 10. Remove the virus and wash twice with serum-free medium 11. Add 2 ml of agarose overlay medium to each well 12. Leave at room temp for 10min and then incubate at 37oC 13. Ready to harvest ~48h post infection 14. Count the number of plaques 15. Fix the cells in 4% PFA for more than 30min 16. Remove the agarose overlay and stain with 2 mg/ml crystal violet 17. Remove the stain and count the number of plaques again Using 1% CMC overlay 18. 3-3.5 ml CMC overlay medium is needed for each well 19. Aliquot serum-free medium to 50 ml falcon 20. Before the end of the 1h incubation, mix 3% CMC and serum-free medium v:v=1:3 21. Remove the virus and wash twice with serum-free medium 22. Add 3-3.5 ml 1% CMC overlay medium to each well 23. Incubate at 37C 24. Ready to harvest ~72-96h post infection 25. Remove the 1% CMC overlay medium 26. Fix the cells in 4% PFA for more than 30min 27. Remove the 4% PFA and stain with 2 mg/ml crystal violet 28. Remove the stain and count the number of plaques

Virus titer = (# of plaques) X (dilution factor) X 5 PFU/ml

Immunofluorescence
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Thaw 4% paraformaldehyde (PFA) Harvest cells in 6-well plate or 35mm dish. Rinse cells briefly in 1-2 ml PBS twice Aspirate PBS, cover with 2 ml 4% PFA. Fix cells for 15-30min at room temperature Aspirate PFA, rinse three times in 1ml PBS for 5min each STOP: Keep the plate/dish at 4C with PBS covering the cells for 1-2 days Cover with 2 ml ice cold methanol, incubate for 10 min at -20oC Prepare (or thaw) blocking solution. Briefly spin Blocking solution: 5% goat serum in 1XPBS/0.3% Triton-X100 Rinse cells in 2 ml PBS for 5min Add 65 l blocking solution to clean flat surface. Invert the cover slip and block for 1h Prepare 1' antibody by diluting with blocking solution. Briefly spin Add 65 l diluted 1' antibody to clean flat surface. Invert the cover slip and probe for overnight at 4C or for 1-2h at room temperature Revert the cover slip and rinse three times in 1 ml PBS for 5min each Prepare 2' antibody by diluting with blocking solution. Briefly spin DAPI (10000X diluted) can be included in the diluted 2' antibody Add 65 l diluted 2' antibody to clean flat surface Invert the coverslip and probe for 1-2h at room temperature in dark Revert the cover slip and rinse three times in 1ml PBS for 5min each Label glass slide. Add 15 l mounting medium to the glass slide. Gently slide the cover slip onto the mounting medium. Squeeze excessive mounting medium until the cover slip no longer move Use nail polish to seal the edge of cover slip Observe under fluorescent microscope or store slides at -20oC

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Total RNA extraction

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Remove medium from dish or well Add 1 ml Trizol to each well of a 6-well plate (or a 35mm dish) Lyse the cells by pipetting up and down several times, transfer to a 1.5ml eppendorf Incubate the homogenized samples at room temp for 5min STOP: Homogenized samples can be stored at -80C for at least 1 month Add 200ul chloroform per 1ml Trizol in the fume hood Shake vigorously by hand for 15sec, stand at room temp for 15min Centrifuge at 12000 x g for 15min at 4C Transfer the clear aqueous phase to a new 1.5ml eppendoff Add 500ul isopropanol and mix by inverting, stand at room temp for 10min STOP: stand at -20C for 1-2h Centrifuge at 12000 x g for 10min at 4C Remove supernatant and wash pellet with 1ml 75% ethanol Centrifuge at 12000 x g for 5min at 4C Repeat 75% ethanol wash STOP: RNA pellet in 75% ethanol can be stored at -20C for at least 1 year Decant the supernatant Briefly spin and completely remove the supernatant Dry pellet in concentrator for 5-10min, do not over dry Add 15-20 l DEPC-water to the pellet Incubate the tube in 55 C heat block for 10-15min Gently flick the tube and spin down Meausre RNA concentration in NanoDrop. Store the RNA at -80C

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RT-PCR
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Thaw reagents and RNA solutions on ice For each template/primer pair, add the following solutions 2-5 g Total RNA in DEPC-water 1 l 10uM oligo-dT or gene specific primer X l DEPC-water Total volume of template/primer mix is 10.5ul Put the tubes to a preheated 70oC heat block for 5min Immediately chill on ice for at least 5min, spin down and keep on ice Prepare the reaction mix by combining the following reagent (per reaction) 4 l 5X reaction buffer 3 l 25mM MgCl2 1 l 10mM dNTP 1 l RT-enzyme 0.5 l RNasin Total volume of reaction mix is 9.5ul per reaction Add 9.5ul reaction mix to each template/primer mix, mix and spin down 25oC for 5min 42oC for 60min 70oC for 15min, cDNA ready for PCR, or stored at -20oC

PCR check clone

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Add 400 l LB broth with proper antibiotic to each of the eppendorf Prepare PCR master mix and aliquot to PCR tubes, (do not include template volume) Use a yellow pippet tip to pick a single colony from the plate Quickly dip the tip into the PCR tube and swirl briefly Press the tip to the corresponding eppendorf and swirl hard to dislodge the colony Generally pick 8-10 colonies. Recover the eppendorf culture at 37C shaker Perform PCR and gel electrophoresis to check for the presense of insert Select 2-3 possitive clones and innoculate the starter for overnight culture

Heat inactivate FBS for culture medium

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Thaw FBS in 37oC water bath (or room temp, may take longer) Warm FBS at 56C for 30min, swirl every 5min to mix Color of FBS may change Aliquot and store at -20C or use to make complete culture medium

6X SDS sample buffer (Final volume 50ml)

1M Tris-HCl pH 6.8 Glycerol SDS (powder) Bromophenol blue

15ml 30ml 6g 30mg

Wear mask when weighting and dissolving SDS powder Disolve SDS in 1M Tris-HCl by heating the solution while stiring Glycerol is viscous, make sure correct volume is used Top up final volume to 50ml Aliquot 950 l to 2ml self stand screw cap tube Add 50 l beta-mercaptoethanol and heat before used

siRNA transfection using Dharmafect (12-well plate)

1. Plate cells with the following density

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H1299 100,000 per well A549 100,000 per well Vero 50,000 per well Thaw siRNA solution (10 M) For each transfection reaction, add 100 l plain medium each to two eppendorf tubes To tube 1, add 5 l siRNA and gently mix To tube 2, H1299 add 2 l DharmaFECT 2 and gently mix A549 2 l DharmaFECT 1 VERO 4 l DharmaFECT 3 Incubate tube 1 and 2 for 5min at room temperature Add the content of Tube 1 to Tube 2 for a total volume of 200 l. Mix by pipetting up and down and incubate for 20min at room temperature

8. Remove culture medium from wells of the 12-well plate 9. Rinse twice with antibiotic free complete medium 10. Add 800 l antibiotic free complete medium to each well 11. Add the 200 l transfection mixture to the appropriate wells drop wise 12. Gently mix by shaking the plate back and forth several times

Final siRNA concentration in the medium is 50 nM Incubate for 24-48 hours (for mRNA analysis) or 72-96 hours (for protein analysis) If necessary, the transfection medium can be replaced with complete medium after 24h A duplicate transfection 24h after the initial transfection may be necessary for VERO