Media, vitamins and so on.

About the recalculation of recipes for the arbitrary volume:

Stock 100ml 150ml 200ml 300ml 600ml 750ml 1. Bactotriptone solid 1g 1.5g 2.0g 3.0g 6.0g 7.5g 2. Bacto Yeast extr. solid 0.5g 0.75g 1.0g 1.5g 3.0g 3.75g 3. NaCl solid 1.0g 1.5g 2.0g 3.0g 6.0g 7.5g 4. H2O mQ 95.3g 143g 190.7g 286g 572g 715g 5. agar top solid 0.65g 0.98g 1.3g 1.95g 3.90g 4.88g 6. agar bot. solid 1.5g 2.3g 3.0g 4.5g 9.0g 11.3g 7. NaOH 10N 16.7µl 25µl 33.4µl 50µl 100µl 125µl 8. MgSO4 1M 1ml 1.5ml 2.0ml 3.0ml 6.0ml 7.5ml

LTB: p.1, 3, 4, 7, 8.
LB: p.1, 2, 3, 4, 7.
LB/Mg: p.1, 2, 3, 4, 7, 8.

• it would be better to mix agar after autoclaving. Otherwise it will solidify as heterogeneous substance (much denser on the bottom).

LB (low salt)

For medium with Zeocine pH 7.0

Stock 100ml 150ml 200ml 300ml 600ml 750ml Bactotriptone solid 1g 1.5g 2.0g 3.0g 6.0g 7.5g Bacto Yeast extr. solid 0.5g 0.75g 1.0g 1.5g 3.,0g 3.75g NaCl solid 0.5g 0.75g 1.0g 1.5g 3.,0g 3.75g H2O mQ 95.3g 143g 190.7g 286g 572g 715g NaOH 10N 16.7µl 25µl 33.4µl 50µl 100µl 125µl

M9

10x

Conc. Stock 50ml 75ml 100ml Na2HPO4x 12 H2O 48mM 358.14g/M 8.60g 12.90g 17.19g KH2PO4 22mM 136.09g/M 1.50g 2.25g 3.00g NaCl 8.5mM 58.44g/M 0.25g 0.38g 0.50g NH4Cl 18.7mM 53/49g/M 0.50g 0.75g 1.00g

M9:

Conc. Stock 100ml 150ml 200ml M9 1x 10x 10ml 15ml 20ml Glucose 0.4% 10% 4ml 6ml 8ml H2O   cтеp. 86ml 129ml 172ml

add additional components:

thiamine 1mM 1M 100µl 150µl 200µl MgSO4 1mM 1M 100µl 150µl 200µl CaCl2 0.1mM 1M 10µl 15µl 20µl

for Petri dishes medium it is necessary to add agar to 3%.

NZY

1 L 200ml NZ amine 10g 2g NaCl 5g 1g Bacto Yeast extr. 5g 1g MgSO4xH2O 2g 0.4g H2O 950ml 190ml

SOC, SOB

(store at NT).

Stock 200ml 400ml Bactotriptone   4.0g 8.0g Bacto Yeast extr.   1.1g 2.2g NaCl 5M 0.4ml 0.8ml KCl 1M 2.0ml 4ml H2O   ~196ml ~391ml

1. after autoclaving to cool to <60^oC;
2. add

0.01V 2M Mg²⁺{= 1M MgCl₂ + 1M MgSO₄}  
0.01V 2M glucose;  

3. 0.2µm filter sterilization;
4. store at -20^oC, (-70^oC) in small aliquots.

SOB - the same solution without glucose.

Terrific Broth

pH 7.0

100ml 150ml 200ml 300ml 667ml Bactotriptone 1.2g 1.8g 2.4g 3.6g 8.0g Bacto Yeast extr. 2.4g 3.6g 4.8g 7.2g 16.0g Glycerol 0.4ml 0.504g 0.6ml 0.757g 0.8ml 1.009g 1.2ml 1.513g 2.667ml 3.362g H2O to V 90ml 135ml 180ml 270ml 600ml add after autoclaving: KPO4, pH7.0 1.0M 10ml 15ml 20ml 30ml 66.7ml

YENB

pH 7.5: (non-salt medium for electro-competent cells preparation).

Conc. Stock 0.45 L 0.5 L 0.9 L 1.0 L Bacto Yeast extr. 0.75% solid 3.4g 3.75g 6.75g 7.5g Nutrient Broth 0.8% solid 3.6g 4.0g 7.2g 8.0g NaOH   10N ~243 µl ~270 µl ~490 µl ~540 µl

2 YT

%(w/w) 200ml 1L Bactotriptone 1.6 3.2g 16g Bacto Yeast extr. 1.0 2g 10g NaCl 0.5 1g 5g H2O 96.9 193.8ml 969ml

Other substances.

IPTG

C₆H₁₈O₅S; Mw=238.3g/M;

Conc. 1ml 5ml 10ml IPTG 200mg/ml 0.2mg 1.0g 2.0g

filter sterilization;

store dry powder at 4^oC (dry),-20^oC (solution in H₂O);

- for plasmids - per 80mm Petri dish ~4µl,
- for M13 - per 3ml of top agar ~1-4µl.

X-Gal

C₁₄H₁₅BrClNO₆ Mw= 408g/M;

Conc. 1ml 5ml 10ml X-Gal 20mg/ml (2%) 20mg 0.1g 0.2g

store dry powder at 4^oC in the dark; solution at -20^oC in the dark (in DMFA (or DMSO with the same result)).

- for plasmids - per 80mm Petri dish 40µl,
- for M13 - per 3ml of top agar 40µl.

Glucose

C₆H₁₂O₆ Mw=180.16;

Conc. Stock 5ml 10ml 50ml 100ml 150ml 200ml C6H12O6 1M 180g/M 0.90g 1.80g 9.01g 18.02g 27.03g 36.03g

filter sterilization.

Maltose.

100x solution for bacteriophage lambda media;

Conc. Stock 10ml 50ml 100ml Maltose 20% solid 2g 10g 20g

filter sterilization.

Thiamine.

Vitamin B₁ Aneurine; C₁₂H₁₇ClN₄OSxHCl; Mw=337.3g/M;

Conc. Stock 1ml 5ml 10ml 25ml C12H17ClN4OSxHCl 1M 337.3g/M 0.337g 1.69g 3.37g 8.43g

filter sterilization.

"Practical Molecular Biology" http://molbiol.edu.ru
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