Methods

Partial purification of lysozyme

1.        For the partial purification of lysozyme, egg whites, carefully separated from the egg yolks, were diluted 3- or 3.3-fold with 0.05 M NaCl solution.

2.       To precipitate the egg  white proteins other than lysozyme, the pH of this mixture was set to 4.0 by carefully adding several drops of 1 N acetic acid and it was diluted with an equal volume of 40% (v/v) ethanol. After 30 minute incubation at room temperature in the presence of ethanol, the mixture is centrifuged at 15,000 x g for 15 min at 4 °C; then the precipitates were discarded.

3.       The supernatants were analyzed for their lysozyme activity and protein content to determine if lysozyme extraction was successful.

4.       A DNA extraction is performed using a kit the epicenter QuickExtract Bacterial DNA Extraction Kit (QEB0905T) and E.Z.N.A. Bacterial DNA Kit from Omega bio-tek (D3350-00) as controls

5.       A DNA extraction is performed using both kits but replacing the kit provided lysozyme with the student prepared egg-white (EW) lysozyme

QuickExtract™ Bacterial DNA Extraction Kit

1. Centrifuge ~108 bacteria at 1,700 x g (5,000 rpm) in a microcentrifuge for 3 minutes to pellet the cells.

2. Wash the bacterial cell pellet once with 0.5 ml of sterile water, then re-centrifuged at 1,700 x g (5,000 rpm) for 3 minutes.

3.Carefully remove and discard the supernatant. Add 100 μl of QuickExtract Bacterial DNA Extraction Solution to the cell pellet.

4. Add 1 μl of Ready-Lyse Lysozyme Solution to each tube and mix gently by inversion. Make certain that both the bacteria and the Ready-Lyse Lysozyme are dispersed in solution, but avoid actions that could cause shearing of the DNA if long gDNA is required.

5 .Incubate the suspension at room temperature for 15 minutes. If the solution is not clearing, wait an additional hour at room temperature. Observe the lysis periodically; digestion can be extended to several hours if necessary. Optional: If it is important to kill any remaining viable bacteria, the sample may be heated at 80°C for 2 minutes

6. The DNA is now ready for PCR, restriction endonuclease digestion, PFGE, or optical mapping.

Note: If the DNA is to be used for PCR, use at full strength or dilute in TE Buffer (10 mM Tris-HCl pH=7.5, 1 mM EDTA). For restriction digests, DNA can be used at full strength or diluted before adding the appropriate 10X restriction endonuclease buffer and enzyme.

Nano Drop Graph

1. Open the app
2. Click “Nucleic Acid”
3. Open arm
4. Add 2 ul of TE Buffer
5. Close arm
6. Click “Blank”
7. Open arm
8. Add 2 ul of bacteria on top of the TE Buffer
9. Close arm
10. Name it
11. Click measure
12. Clean off
13. Repeat for as many samples you have