Plasmid Isolation Using Alkaline Lysis

Plasmid Isolation Protocol

1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial colony. The tube was incubated at 37 ˚C overnight with vigorous shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated gentle inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After the addition of 2ul RNase A (10mg/ml), the mixture was incubated for 20 minutes at room temperature to remove RNA. 

Note:

• Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage.
• Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.
• Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells. 
• Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). 
• Three things happen during this stage: 

1. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo, and toothpaste.
2. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.
3. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

• Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things:

1. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single-stranded DNA (ssDNA).
2. The ssDNA is precipitated since large ssDNA molecules are insoluble in high salt.
3. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from your plasmid DNA. 

Now that you've made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS, and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

• Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt mask the charges and allows DNA to precipitate. This will place your DNA in the pellet.
• Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.
• Resuspend your now clean DNA pellet in the buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution. 

DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you'll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification. 

Hemagglutination (HA) Assay Protocol

The hemagglutination assay is a method for titering influenza viruses based on their ability to attach to

molecules present on the surface of red blood cells. A viral suspension may agglutinate the red blood

cells, thus preventing them from settling out of suspension. By serially diluting a virus in a 96-well plate and adding a consistent amount of red blood cells, an estimation of the amount of virus present can be made. 

Equipment and Materials Required

• Certified Biological Safety Cabinet
• Tabletop centrifuge with appropriate fittings
• Inverted microscope (optional)
• 15 ml conical tubes
• Disposable pipettes – 1 ml, 5 ml, 10 ml
• Micropipette and sterile disposable aerosol resistant tips – 160 µl
• PBS
• Turkey red blood cells in Alsevers solution purchased from a supplier such as Lampire Biological Products
• round-bottomed 96-well dish 

Turkey RBC preparation:

1. 4 ml of turkey blood is pipetted into a 15 ml conical and topped off with PBS.
2. Spin in a tabletop centrifuge at 800 rpm for 10 minutes.
3. Aspirate the supernatant without disturbing the blood cells.
4. Add 12 ml PBS and mix by inverting – do not vortex.
5. Spin at 800 rpm for 5 minutes and repeat wash two more times.
6. Aspirate supernatant after final wash and add enough PBS to make a 10% solution of red blood cells. This solution is useable for one week.
7. Make a final working solution of 0.5% RBCs in PBS. 

Viral Dilution and Assay:

1. A round-bottomed 96-well dish is preferred for this assay. Flat-bottomed plates will also work but need to be placed at an incline to develop.
2. To each well, add 50 μl PBS.
3. In the first column, add 50 µl of virus sample.
4. Mix each well and transfer 50 µl to the next well on its right. Repeat mixing and transferring 50µl down the length of the plate. Discard 50 µl from the last well into a bleach solution.
5. Add 50 µl of 0.5% red blood cell working solution to each well. Mix gently.
6. Leave at room temperature for 30-60 minutes to develop. Negative results will appear as dots in the center of round-bottomed plates. Positive results will form a uniformly reddish color across the well.
7. The virus’s HA titer is a simple number of the highest dilution factor that produced a positive reading.