Biomod/2014/NTU/Protocol

From OpenWetWare

Jump to: navigation, search

Supplement

Protocol

start

Folding Scaffold

Materials:

1. 1x Buffer (5 mM Tris, 1 mM EDTA and 10 mM MgCl2, pH 7.8) 2ul
2. Staple 100nM (in buffer) 3ul
3. Scaffold 10nM (in buffer) 6ul
4. water
total: 20ul

Steps:

1. Mix scaffold and staples together.
2. 94C to 86C at 4C per 5 minutes;
&nbsp&nbsp&nbsp 85C to 70C at 1C per 5 minutes;
&nbsp&nbsp&nbsp 70C to 40C at 1C per 15 minutes;
&nbsp&nbsp&nbsp 40C to 25C at 1C per 10 minutes.

Preparing/Running an Agarose Gel

Materials:

1. Running buffer (11 mM MgCl2 in 0.5X TAE)
2.Loading Dye(10X)
3.Gel (1% agarose, contain 11mM MgCl2)
4. 0.5X TAE (5X TAE: 0.45M tris, 0.45M acetic acid, 10mM EDTA)

Gel preparation

1.An agarose 1% solution was prepared with TAE 1X buffer.
2.Heat the solution until agarose is dissolved totally and then cool it down.
3.Pour the solution into the mold and put the comb at the correct site. Wait for 30 minutes so that the gel can solidify.

Sample loading

Samples were mixed with the loading dye in a ratio of 1:10.

Gel running and staining

Gels were run for 40 minutes on the ice to prevent it from demaged by the high temperature, using 120V, and then stained with Ethidium bromide for 10 minutes for UV visualization.

H5N2 virus & HA test

Materials

Influenza virus stock
Egg diluent: antibiotic-supplemented KPBS or tryptose phosphate broth
10- to 11-day-old embryonated chicken eggs
70% ethanol
Glue (Elmers), nonsterile
22-G, 1 1/2-in. and 18-G, 1/2-in. needles
1-ml syringe
33°Cto 35°C incubator
Forceps, sterile
10-ml pipets
50-ml plastic concical tubes
2-ml cryovials, sterile (Nunc)

Egg inoculation

1. Dilute influenza virus stock in egg diluent.
2. Arrange three eggs with the airsac up. Spray the surface of eggs with 70% ethanol.
3. Punch a small hole in the shell over the air sac using an 18-G, 1/2-in. needle and
dispose of the needle in an appropriate sharps container.
4. Aspirate 0.6 ml diluted virus sample into a 1-ml syringe with a 22-G, 11/2-in. needle.
5. Insert the needle at a 45° angle into the allantoic cavity and inoculate 0.2 ml influenza
virus dilution; repeat with the other eggs.
6. Discard syringe and needle into a sharps safety container.
7. Seal the hole punched in the eggshell with a drop of glue.
8. Incubate human influenza A virus for 48 hr at 33°to 35°C.

Harvest virus from infected eggs

9. Chill eggs overnight (8 to 24 hr) at 4°C to halt embryo viability and minimize the
flow of blood into the allantoic fluid during harvest.
10. With sterile forceps, break the shell over the air sac and push aside the allantoic
membrane with the forceps, taking care not to break the yolk.
11. Using a 10-ml pipet, aspirate the allantoic fluid and place in a labeled 50-ml plastic
conical tube. Dispose of the eggs in an appropriate biological waste container.
12. Centrifuge tubes 5 min at 500×g, 4°C, to pellet any blood cells and tissue fragments.
Transfer supernatant fluid into fresh 50-ml tube. Keep on ice (4°C).
13. Perform a hemagglutination test
14. Dispense allantoic fluid into 2-ml aliquots in 2-ml sterile cryovials and store at -7°to -80°C

Quantification of H5N1 virus by hemagglutination assay

Materials

Phosphate-buffered saline containing potassium
Influenza virus stock (e.g., allantoic or amniotic fluid)
Standardized turkey red blood cells
96-well V-bottom microtiter plate

Steps

1. Pipet 50 ul PBS into wells 2 through 12 across a 96-well V-bottom plate
2. Pipet 100 ul influenza virus stock into the first column of the 96-well plate.
3. Perform a two-fold dilution series across the 96-well plate by transferring 50 ul between wells, disposing of the final 50 ul from the last well.
4. Add 50 ul standardized turkey red blood cells to all wells. Tap the plate gently to mix.
5. Incubate 96-well plate for 30 min at room temperature (24°to 27°C).
6. Observe endpoint of agglutination and record titer per 50 ul of sample
7. Store virus stocks with desired HAU titers .Dispose of materials in an appropriate biological waste container.

Deacivating H5N2 virus

Treat the virus with Beta propyl lactone on a shaker for 3 days.

Personal tools