Category Archives: Bacteria

Bacterial DNA extraction

1. In a biosafety cabinet, transfer 1000uL of liquid bacterial culture from a 15mL conical tube to a microcentrifuge tube. Return the conical tube to 4˚C.
2. Spin the microcentrifuge tube for 5 minutes at 10,000g to pellet the bacterial cells. Discard supernatant.
3. Resuspend the pellet in 150uL Tissue & Cell Lysis Solution (Epicentre), and add 10uL Proteinase K at 20 mg/mL (Qiagen). Incubate at 55˚C for 30 minutes.
4. Add 2 uL of RNase A at 5 ug/uL (Epicentre), and incubate at 37˚C for 60 minutes, followed by 5 minutes on ice.
5. Add 75uL MPC Protein Precipitation Reagent (Epicentre) and vortex for 10 seconds. Centrifuge at 12,000g for 10 minutes. Note: following this centrifugation, the protein pellet will be entirely transparent. Be sure to orient the tubes in the centrifuge such that the pellet will be in a predictable location.
6. Transfer supernatant to a fresh microcentrifuge tube and add 250uL isopropanol to the supernatant.
7. Mix tubes by inverting 10 times, and incubate at room temperature for 20 minutes.
8. Centrifuge at 12,000g at 4˚C for 10 minutes. Discard supernatant without disturbing the pellet.
9. Add 500uL 70% ethanol and centrifuge at 12,000g at 4˚C for 5 minutes. Discard supernatant.
10. Repeat step 9.
11. Remove as much ethanol from the tube and dry the tube with the cap open on the bench for 20 minutes, or until entirely dry.
12. Resuspend the DNA pellet with 50 uL TE buffer. Incubate for 5 minutes at 55˚C to resuspend the pellet.
13. Vortex briefly and spin down. Store at -20˚C.

LB Media Protocol

FOR 600mL OF LB BROTH MEDIA:

1. Weigh out 6 grams of Tryptone

2. Weigh out 3 grams of yeast extract

3. Weigh out 6 grams of NaCl

(3.5). FOR LB AGAR MEDIA: Weigh out 9 grams of agar

4. Combine all of the necessary substances into a 1L bottle

5. Measure out 600mL of distilled water into a graduated cylinder and pour into the bottle

6. Tighten the cap, Mix, Perform the quarter turn for ventilation, tape the cap to the bottle with autoclaving tape, and label the contents of the bottle; the bottle is now ready for autoclaving.

 

 

TY Recipe

TY (Tryptone Yeast)

Ingredient, g/L, g/600mL
tryptone, 6, 3.6
yeast extract, 3, 1.8
CaCl2, 0.38, 0.228
Dissolve first 3 ingredients in dH2O. If making solid media, add agar and **DO NOT MIX***—the autoclave will melt the agar for proper mixing.
bacto-agar, 16, 9.6
Autoclave on liquid 30min cycle (no exhaust). Remove promptly to a water bath: 50C will keep everything liquid; ~40C [hot bath] is the temperature you want to pour at.

NOTES:
use dH2O from the tap
dissolve before autoclaving to avoid crystals
for broth (liquid media), omit agar