Medium &Reagents


  1. LB medium
  2. LB-plate
  3. SOB medium
  4. SOC medium
  5. TB

LB medium for 1l

Bacto Tryptone 10g

Bacto Yeast Extract 5g

NaCl 5g

5N NaOH 0.2ml (this brings pH to 7.0)

Make up to 1l with dH2O and sterilize by autoclaving

LB plate (c.a. 20ml for one plate)

for 1l

for 500ml

for 200ml

Bacto Tryptone

10g

5g

2g

Bacto Yeast Extract

5g

2.5g

1g

Bacto-Agar
(or INAAGAR BA-30 )

15g
10-12g

7.5g
5-6g

3g
2-2.4g

NaCl

5g

2.5g

0.5g

5N NaOH

0.2ml

0.1ml

0.04ml

dH2O

Make up to 1l

sterilize by autoclaving

　

Appropriate antibiotics can be added after the solution cooling down under 60｡C.

　

SOB medium

　

Bacto Tryptone 20g(2.0%)

Bacto Yeast extract 5g(0.5%)

5M NaCl 2ml(10mM)

2M KCl 1.25ml(2.5mM)

miliQ water 990ml

make up to 1000ml

sterilize by autoclaving

　

add 10ml of 2M Mg2+solution just before use (1M MgSO4 7H2O +1M MgCl2 6H2O)

　

SOC medium

Bacto Tryptone 20g

Bacto Yeast Extract 0.5g

NaCl 58.4mg (final 10mM)

KCl 18.6mg (final 2.5nM)

Make up to 93ml with dH2O and sterilize by autoclaving

Wait until it cool down to room pemperature and add

1M MgCl2 1ml

1M MgSO4 1ml

0.4M glucose 5ml

TB (transformation buffer)

　

PIPES 3.0g(10mM)

CaCl2 2H2O 2.2g(15mM)

KCl 18.6g (250mM)

add 950ml dH2O

at low pH solution will be milky

　

bring pH to 6.7 -6.8 with 5N KOH

5N KOH (14.03g/50ml KOH m.w. :56.11)

add 10.9g of MnCl2 4H2O

　

make up to 1l

sterilize by filtration (Do not autoclave!)

Store at 4｡C

Antibiotics concentration

　

Stock

Final

Ampicillin

200mg/ml (2000x)

100ug/ml

water

Kanamycin

20mg/ml (1000x)

20ug/ml

water

Tetracyclin

5mg/ml (250x)

20ug/ml

Ethernol

　

　

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