IIT Madras

And then the E.coli said, "Let there be light"

Protocols

For 5 ml Cultures: 12 – 16 hours incubation.
For 50 ml cultures: 8 hours of primary (2ml) and 16 hours of secondary (50 ml) add 50 ul.

• Pellet down the cells at 12000 rpm for 2 min. Use same eppendorf for 5 ml cultures. Remove all the media from pellet.
• Add ice cold Soln 1, 250 ul, Vortex well. Incubate in ice for 5 min.
• Add fresh soln II, 250 ul. Invert mix. Incubate at Room temperature for 5 min.
• Add ice cold soln III, 250 ul. Invert mix. Incubate on ice for 5 min.
• Add RNase, and incubate at 43 C for half an hour.
• Add equal volume of Phenol Chloroform. Vortex well. Centrifuge at 12000 rpm for 10 minutes. Take the supernatant.
• Add 0.6 volumes (~450 ul) of isopropanol. Shake well. Incubate at room temperature for 10 minutes. Centrifuge at 12000 rpm 4 C. 10 minutes.
• Discard isopropanol. Wash pellet with 70% ethanol. Spin and Drain Ethanol.
• Let tubes dry at 37 C till the eppendorfs don’t smell of ethanol.
• Add 20 ul – 50 ul autoclaved MilliQ wateror (10mM Tris+1mM EDTA) with RNase. Leave for 30 mins at 43 C if RNase is added.

Reagents

Lysis Buffer 1

• 50mM -- Glucose
• 25mM -- TRIS-Cl (pH 8.0)
• 10mM -- EDTA (pH 8.0)

Autoclave and store at 4 C

Lysis Buffer 2

• 8ml -- Water
• 1ml -- 10% SDS
• 1ml -- 2N NaOH

Lysis Buffer 3 (for 100ml)

• 60ml -- 5M Sodium Acetate
• 11.5ml -- Glacial Acetic Acid
• 28.5ml -- Water

Check pH. It should/will be around 5.2-5.4. Autoclave & store at 4 C.

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