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M9-Minimal-Media

1x M9 salts                                             100 mL/L 10x M9 salts

0.4% (w/v) glucose                               20 mL/L 20% glucose

1 mM MgSO 4                                         1 mL/L 1 M MgSO 4

0.3 mM CaCl 2                                                                      300 µL/L 1 M CaCl 2

1 µg biotin                                              1 ml/L Biotin (1 mg/ml)

1 µg thiamin                                          1 ml/L Thiamin (1 mg/ml)

Trace Elements                                     60 µL/L Trace Elements Stock                                     

( 5 g/L Yeast Extract                               50 ml/L Yeast Extract Stock 100 g/L use for adaption to pure M9 )

sterile deionized H 2 O                           up to 1 L

                                                                                              

• Prepare Stock Solutions

 

M9 salt solution (10X)

Na 2 HPO 4 -2H 2 O 75.2 g/L

KH 2 PO 4 30 g/L

NaCl 5 g/L

NH 4 Cl 5 g/L

Dissolve the salts in 800 ml deionized H 2 O and adjust the pH to 7.2 with NaOH. Add deionized H 2 O to a final volume of 1 L and autoclave for 15 min at 121°C.

20% Glucose stock solution

Glucose*H 2 O 220 g/L

Add deionized H 2 O to a final volume of 1 L and autoclave for 15 min at 121°C.

1M MgSO 4 stock solution

MgSO 4 *7H 2 O 24,65 g/100 ml

Add deionized H 2 O to a final volume of 100 ml and autoclave for 15 min at 121°C.

1M CaCl 2 stock solution

CaCl 2 *2H 2 O 14,70 g/100 ml

Add deionized H 2 O to a final volume of 100 ml and autoclave for 15 min at 121°C.

Biotin (1 mg/ml)

Dissolve 50 mg biotin in 45 ml deionized H 2 O. Add small aliquots of 1M NaOH until the biotin has dissolved. Add deionized water to a final volume of 50 ml. Sterilize the solution over a 0.22 µm filter. Prepare 1 ml aliquots and store at -20°C.

Thiamin (1 mg/ml)

Dissolve 50 mg thiamin-HCl in 45 ml deionized H 2 O. Add deionized water to a final volume of 50 ml. Sterilize the solution over a 0.22 µm filter. Prepare 1 ml aliquots and store at -20°C.

Trace elements stock solution                       

FeSO 4 ·7 H 2 O                                             40.0 g/L

MnSO 4 ·H 2 O                                               10.0 g/L

AlCl 3 ·6 H 2 O                                                10.0 g/L

CoCl 2 ·6 H 2 O                                                 7.3 g/L

ZnSO 4 ·7 H 2 O                                               2.0 g/L

hNa 2 MoO 4 ·2 H 2 O                                         2.0 g/L

CuCl 2 ·2 H 2 O                                                 1.0 g/L

H 3 BO 3                                                           0.5 g/L

37% (12 M) HCl conc.                           414 mL/L

 

Dissolve in HCl diluted 1:1 with deionized H 2 O and fill up to 1 L with deionized H 2 O; the final solution is 5 M HCl and can be stored indefinitely at RT. Sterilize the solution over a 0.22 µm filter before use.

Yeast extract Stock (100 g/L)

Yeast extract 100 g

Add deionized H 2 O to a final volume of 1 L. Sterilize the solution over a 0.22 µm filter.

 

• Mix the components in a sterile bottle.

 

Adjusting the pH of M9- and LB-media

Adjust the pH of M9 media from ~7,4 to 8,5

add 0,68ml of 2M NaOH to 100ml M9 (be carefull, unknown prezipitates appear)

 

Adjust the pH of M9 media from ~7,4 to ~6
add 4,68ml of 2M HCl to 100ml M9

 

Adjust the pH of LB media from pH ~7 to pH 8,5

add 0,66ml of 2M NaOH to 100ml  LB

 

Adjust the pH of LB media from pH ~7 to 6,0
add 0,575 ml of 2M HCl to 100ml  LB

PPPPPPPPPPHHHHHHHHHHHHHHHHH

 

Electro-competent cells

• inoculate single colony from a fresh plate 10 mL ONC (in LB)

• 500 mL LB main culture (5 mL ONC)

• growth (@30°C) until OD 0.35-0.4
• chill on ice 10-20 min
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend (carefully!) in 2x 400 mL ice-cold water
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend (carefully!) in 400 mL ice-cold 10% glycerol
• centrifuge (4000 rpm, 10 min, 4°C)
• resuspend in 10 mL 10% glycerol
• prepare 40 µL aliquots (on ice!)
• shock-freeze them in liquid nitrogen
• store @ -80°C (up to 6 months)

 

Transformation (electro-competent cells)

• thaw cells on ice
• add 1-2 µl DNA (up to 2 ng) or 2-10 µl Ligationmix (on ice!!)
• transfer cells into a cuvet
• electro-shock
• add 750-900 µl LB- or SOC-media
• resuspend carefully by pipetting up and down
• tranfer to an new steril tube
• regenerate for 45 min at 37°C and shake at 300 rpm
• plate on LB-plate (with anitbiotic)
• incubate over night at 37°C
• check for colonys

Transformation (chemically competent cells for Gibson Cloning)

• thaw cells on ice
• add 2 µl DNA (on ice!!)
• place micture on ice for 30 min
• heat-shock at 42°C for 1 min (thermomixer)
• transfer on inc for 2 min
• add 750-900 µl LB- or SOC-media
• regenerate for 45 - 60 min at 37°C and shake at 300 rpm
• plate on LB-plate (with anitbiotic)
• incubate over night at 37°C
• check for colonys

PCR with Q5-polymerase with proofreading activity from NEB

 

X µl template (up to 1 ng)

       2.5 µl fw_primer (10 µM)

       2.5 µl rev_primer (10 µM)

       0.5 µl Q5-Polymerase

10 µl Q5-Buffer 5x

10 µl GC-Enhancer (additional)

1 µl dNTP´s (10 mM)

26.6 - X µl ddH 2 0

50 µl in total