"

From 2010.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

'''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''' #Grow 4 mL cultures of your bacteria to late log phase in nutrient broth. #Prepare two tubes of 1 mL of the bacteria culture in Eppendorf tubes and centrifuge at 10,000 × g for 10–15 minutes. #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. #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. #Incubate both samples at room temperature for 1 hour, mixing every 15 minutes. #Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes. #Resuspend the pellets in 1 mL of 0.85% NaCl and centrifuge again as in step 1.6. #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). #Use live and dead bacteria at your desired concentration for staining experiments shown below. '''Staining Bacteria in Suspension''' #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. #For each 100 uL of your bacteria sample and live and dead bacteria control suspensions, add 1 uL of the dye mixture. #Mix thoroughly and incubate at room temperature in the dark for 15 minutes. #Trap 5 µL of the stained bacterial suspension between a slide and an 18 mm square coverslip. #Observe under a fluorescence microscope equipped with any of the filter sets as below. '''Selection of Optical Filters''' <br /> <u>Longpass and dual emission filters useful for simultaneous viewing of DMAO and EthD-III stains</u> <br /> Omega Filters: XF25, XF26, XF115 <br /> Chroma Filters: 11001, 41012, 71010 <br /> <u>Bandpass filters for viewing DMAO alone</u> <br /> Omega Filters: XF22, XF23 <br /> Chroma Filters: 31001, 41001 <br /> <u>Bandpass filters for viewing EthD-III alone</u> <br /> Omega Filters: XF32, XF43, XF102, XF108 <br /> Chroma Filters: 31002, 31004, 41002, 41004 <br /> Adapted from: http://www.openwetware.org/images/b/b9/Cell_Death_Assay.pdf

Return to LIVE/DEAD® BacLight - Bacterial Viability Kit.