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From 2010.igem.org

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Revision as of 15:37, 29 June 2010

Kit Components

• Component A, DMAO: 2x100 µL, 5 mM in DMSO
• Component B, EthD-III: 2x150 µL, 2 mM in DMSO

Preparation of Live and Dead Bacterial Suspensions as Controls

1. Grow 4 mL cultures of your bacteria to late log phase in nutrient broth.
2. Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes.
3. Remove the supernatant and resuspend the pellet of one tube in 0.3 mL of 0.85% NaCl solution and another tube in 1 mL of 0.85% NaCl.
4. Add 0.7 mL isopropyl alcohol into the tube with 0.3 mL of 0.85% NaCl and mix well (final concentration of isopropyl alcohol: 70%) for preparing dead bacteria.
5. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes.
6. Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes.
7. Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6.
8. Determine the optical density at 670 nm (OD670) for a 3 mL aliquot of the bacterial suspensions in glass or acrylic absorption cuvettes (1 cm pathlength).
9. Use live and dead bacteria at your desired concentration for staining experiments shown below.

Staining Bacteria in Suspension

1. Combine one volume of Component A and two volumes of Component B in a microcentrifuge tube, mix thoroughly and add 8 volumes of 0.85% NaCl solution to derive 100X dye solution.
2. For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture.
3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
4. Trap 5 µL of the stained bacterial suspension between a slide and an 18 mm square coverslip.
5. Observe under a fluorescence microscope equipped with any of the filter sets as below.

Selection of Optical Filters
Longpass and dual emission filters useful for simultaneous viewing of DMAO and EthD-III stains
Omega Filters: XF25, XF26, XF115
Chroma Filters: 11001, 41012, 71010
Bandpass filters for viewing DMAO alone
Omega Filters: XF22, XF23
Chroma Filters: 31001, 41001
Bandpass filters for viewing EthD-III alone
Omega Filters: XF32, XF43, XF102, XF108
Chroma Filters: 31002, 31004, 41002, 41004

Adapted from: http://www.openwetware.org/images/b/b9/Cell_Death_Assay.pdf