From 2014.igem.org

Revision as of 18:13, 17 October 2014

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2^nd June

• We use DH5α E.coli for cloning and others manipulations


• Terrific Broth : 1L
  
  

  Deionized H2O	to 900 mL
  Bactotryptone	12 g
  Yeast extract	24 g
  Glycerol 10%	4 mL

  
  
  Shake until the solutes have dissolved and then sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Allow the solution to cool to 60°C or less, and then add 100 mL of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4.
  (This solution is made by dissolving 2.31 g of KH2PO4 and 12.54 g of K2HPO4 in 90 mL of H2O. After the salts have dissolved, adjust the volume of the solution to 100 mL with H2O and sterilize by autoclaving for 20 min at 15 psi [1.05 kg/cm2] on liquid cycle.)



• SOB : 1L
  
  

  2%	Bactotryptone	20g
  0,5	Yeast Extract	5g
  10mM	NaCl	2mL (5M stock)
  2.5mM	KCl	2.5mL (1M stock)
  10mM	MgCl2	10mL (1M stock)
  10mM	MgSO4	10mL (1M stock)

  
  
  pH media to 7 with NaOH, then autoclave.
  
  
  Transformation Broth (TB): 1L
  
  

  10 mM	Pipes	3.02 g
  15 mM	CaCl2	2.21 g
  250 mM	KCl	18.64 g

  
  
  Mix Pipes, CaCl2 and KCl in 900mL of Millipore water. Add NaOH until pH 6.7 (Solution become uncolored). Add MnCl2 (See below), stir, adjust volume to 1L, then filter sterilize. Store at 4°C.
  
  

  55 mM

  MnCl2

  10.89 g

  
  
  DAY ONE : Grow 12 mL overnight culture of favorite strain of E.Coli in 2XTY (~LB) (We use a select gold Efficiency, which are comparable to DH5α according to Bioline). Make SOB and TB.