• Autoclave 0.1 M CaCl2 in 20% glycerol.
• Primary culture in 5 ml of LB broth for 16 hours.
• Add 1 ml of primary culture into 50 ml of media for secondary culture.
  Check OD at 600 nm.
• Incubate secondary cultures at 37 C in a shaker.
• Check OD600 after 1 hour, 1.5 hours, 2 hours.
• Keep the cells on ice (for 10 minutes) for harvesting as soon as OD600 reaches 0.4 (0.4 -0.6 is the range).
• Pellet down the cells at 6000 rpm for 10 min (4 C ).
  (Each 50 ml culture can be added to 50 ml falcon tubes)
• Add 10 ml of ice cold 0.1M CaCl2.
• Re-suspend the pellet and keep on ice for 10 minutes.
• Centrifuge at 6000 rpm for 10 min (4 C).
• Re-suspend the pellet in 5 ml ice cold 0.1 M CaCl2 Solution.
• Again, keep on ice for 10 min. and centrifuge at 6000 rpm for 10 min.
• . Re-suspend the pellet in 2 ml ice cold 0.1 M CaCl2 Solution. Alliquot 100 ul into a number of tubes and store them at -80 C.

• Add 2 ul of Plasmid DNA to competent cells (100 ul aliquot) on ice for 30 minutes.
• Heat shock : 90 s for 43 C.
• Keep on ice for 2 minutes.
• Add 900 ul of LB broth (sterile) into each microfuge tubes.
• Incubate in shaker at 37 C for 1 hour.
• Plate on plates (with antibiotics if necessary) and incubate at 37 C overnight.

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.

• Prepare a 0.8% low melthing agarose gel
• Add 50 ul preparative reaction product into large wells
• Run the gel at 100 V for 30 – 45 mins
• Cut the gel with restricted DNA and keep it in an eppendorf
• To the cut DNA, add 3 volumes of Chaotropic salt. Incubate at 50 C for 10 minutes
• Add 10 ul of GPS (Glass Powder Solution) for a PCR product and 15 ul for a vector and incubate at Room Temperature for 10 mins. Shake the tubes continuously so that GPS is in suspension and doesn’t settle down.
• Centrifuge the tubes at 12000 rpm for 2 minutes and discard supernatant.
• Wash pellet by adding 50 volumes of wash buffer. That is, 500 ul of Wash buffer for insert and 750 ul for vector.
• Resuspend the pellet in 20 ul of water and heat at 50 C for 10 minutes. Centrifuge the tubes at 12000 rpm for 2 mins. Store supernatant at -20 C.