Working Protocols
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
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