Working Protocols

M9-Minimal-Media

Concentration	add
1x M9 salts	100 mL/L 10x M9 salts
0.4% (w/v) glucose	20 mL/L 20% glucose
1 mM MgSO4	1 mL/L 1 M MgSO4
0.3 mM CaCl2	300 µL/L 1 M CaCl2
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
sterile ddH2O	add to a final volume of 1 L
5 g/L Yeast Extract	add 50 ml/L Yeast Extract Stock 100 g/L for adaption to pure M9

Prepare Stock Solutions:

M9 salt solution (10X)

Na2HPO4 * 2 H2O 75.2 g/L

KH2PO4 30 g/L

NaCl 5 g/L

NH4Cl 5 g/L

Dissolve the salts in 800 ml ddH2O and adjust the pH to 7.2 with NaOH. Add ddH2O to a final volume of 1 L and autoclave for 15 min at 121°C.

20% Glucose stock solution

Glucose * H2O 220 g/L

Add ddH2O to a final volume of 1 L and autoclave for 15 min at 121°C.

1M MgSO4 stock solution

MgSO4 * 7 H2O 24.65 g/100 ml

Add ddH2O to a final volume of 100 ml and autoclave for 15 min at 121°C.

1M CaCl2 stock solution

CaCl2 * 2 H2O 14.70 g/100 ml

Add ddH2O 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 ddH2O. Add small aliquots of 1M NaOH until the biotin has dissolved. Add ddH2O 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 ddH2O. Add ddH2O 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

FeSO4 * 7 H2O 40.0 g/L

MnSO4 * H2O 10.0 g/L

AlCl3 * 6 H2O 10.0 g/L

CoCl2 * 6 H2O 7.3 g/L

ZnSO4 * 7 H2O 2.0 g/L

Na2MoO4 * 2 H2O 2.0 g/L

CuCl2 * 2 H2O 1.0 g/L

H3BO3 0.5 g/L

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

Dissolve in HCl diluted 1:1 with ddH2O and fill up to 1 L with ddH2O; 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 ddH2O to a final volume of 1 L. Sterilize the solution over a 0.22 µm filter.

Mix all components in a sterile bottle.

Adjusting the pH of M9- and LB-media

to adjust the pH of M9 media from 7.4 to 8.5, add 0.68ml of 2M NaOH to 100 ml M9 (be carefull, unknown precipitates appear)

to adjust the pH of M9 media from 7.4 to 6, add 4.68ml of 2M HCl to 100 ml M9

to adjust the pH of LB media from pH 7 to pH 8.5, add 0.66ml of 2M NaOH to 100ml LB

to adjust the pH of LB media from pH 7 to 6.0, add 0.575 ml of 2M HCl to 100 ml LB

LPM-media

50 mM Tris base

20 mM KCl

7.5 mM (NH4)2SO4

240 µM MgCl2

18.5 µM CaCl2

6.5 µM FeCl3

0.2% Glucose

0.6% Bacto peptone

buffer with 0.1 M MOPS for pH-range 7 to 8.5, adjust pH with NaOH /

buffer with 0.1 M MES for pH 5 to 4, adjust pH with HCl

fill up to final volume with ddH2O

Electro-competent cells

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

500 mL LB main culture (5 mL ONC)

growth (at 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 at -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
transfer to an new steril tube
regenerate for 45 min at 37°C and shake at 300 rpm
plate on LB-plate (with antibiotic)
incubate overnight at 37°C
check for colonies

Transformation (chemically competent cells for Gibson Cloning)

thaw cells on ice
add 2 µl DNA (on ice!)
place mixture 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 antibiotic)
incubate overnight at 37°C
check for colonies

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 ddH20

50 µl in total

		25 -30 Cycles
	Stage1	Stage2			Stage3
	Initial Denaturation	Denaturation	Annealing	Elongation	Final Elongation
Temperature	98°C	98°C	Lowest Tm -3°C	72°C	72°C
Time	30 s	5-10 s	10-30 s	1 kb/20-30 sec	2 min

PCR with GoTaq-polymerase without proofreading activity from Fermentas

X µl template (< 0.05 µg/10 µl)

0.5 µl fw_primer (10 µM)

0.5 µl rev_primer (10 µM)

0.4 µl MgCl2

0.2 µl dNTP´s (10 mM)

0.05 µl GoTaq-Polymerase

2 µl Polymerasepuffer 5xGreen

6.35 - X µl ddH20

10 µl in total

		25 -30 Cycles
	Stage 1	Stage 2			Stage 3
	Initial Denaturation	Denaturation	Annealing	Elongation	Final Elongation
Temperature	95°C	95°C	Lowest Tm	72°C	72°C
Time	2 min	30 - 60 sec	30 - 60 sec	1 kb/1 min	5 min

Gibson-Cloning (NEBuilder HiFi DNA Assembly Cloning Kit)

Set up the following reaction on ice:

2 – 3 fragments: vector:insert = 1:2 (use 50 – 100 ng of vector)

X µl total amount of fragments (0.03 – 0.2 pmol)

10 µl NEBuilder HiFi DNA Assembly Master Mix

10 - X µl ddH20

20 µl in total

4 – 6 fragments: vector:insert = 1:1 (use 50 – 100 ng of vector)

X µl total amount of fragments (0.2 – 0.5 pmols)

10 µl NEBuilder HiFi DNA Assembly Master Mix

10 - X µl ddH20

20 µl in total

Incubate sample at 50°C for 15 min for 2 – 3 fragments or 60 min for 4 – 6 fragments
Transform 2 µl of the assembly reaction into competent cells

Restriction Digest (Fast Digest Enzymes from ThermoScientific)

set up following reaction:

1 µl enzyme (XbaI, EcoRI, PstI, BcuI)

2 µl FastDigest Green Buffer 10x

X µl DNA (plasmid DNA up to 1 µg or ~0.2 µg PCR product)

17 - X µl ddH0

20 µl in total

incubate for at least 15 min at 37°C
inactivate for 5 min at 80°C or purify with PCR-purification kit

Ligation

1 µl T4-Liagse

2 µl T4 Ligase Buffer 10x

X µl insert DNA (1:1 molar ratio over vector)

Y µl vector DNA (20 – 100 ng)

17 – (X+Y) µl ddH20

20 µl in total

incubate for at least 15 min at 17 - 22°C
inactivate for 10 min at 65°C or purify with PCR-purification kit
ready for transformation

Miniprep (Wizard® Plus SV Miniprep DNA Purification System from Promega)

pellet 1 – 10 ml ONC
resuspend pellet with 250 µl Cell Resuspension Solution
add 250 µl Cell Lysis Solution and invert to mix
add 10 µl Alkaline Protease Solution and invert to mix
incubate for 5 min at RT
add 350 µl Neutralizations Solution and invert to mix
centrifuge at top speed for 10 min
insert Spin Column into Collection Tube
decant cleaned lysate into Spin Column
centrifuge at top speed for 1 min
discard flow through
add 750 µl Wash Solution
centrifuge at top speed for 1 min
discard flow through
add 250 µl Wash Solution
centrifuge at top speed for 5 min
discard flow through
centrifuge for another 5 min to get rid of ethanol
transfer Spin Column to a new reaction tube
add 20 – 50 µl nuclease free water
centrifuge at top speed for 1 min

Miniprep (Wizard® SV Gel and PCR Clean-Up System from Promega)

A: Dissolving a gel slice

transfer gel into a reaction tube
add 10 µl Membrane Binding Solution per 10 mg gel slice
Incubate at 50 – 65°C until the gel is completely dissolved
Vortex

B: Processing PCR products

Add an equal volume of Membrane Binding Solution

Insert Spin Column into Collection Tube
Transfer dissolved gel mixture or prepared PCR product into the Spin Column
Incubate for 1 min at RT
Centrifuge at top speed for 1 min
Discard flow through
Add 700 µl Washing Solution
Centrifuge at top speed for 1 min
discard flow through
add 500 µl Wash Solution
centrifuge at top speed for 5 min
discard flow through
centrifuge for another 5 min to get rid of ethanol
transfer Spin Column to a new reaction tube
add 20 – 50 µl nuclease free water
incubate for 1 min at RT
centrifuge at top speed for 1 min

Cultivation of Escherichia coli in M9-Minimal-Media

Add appropriate selection marker to all media involved.

Pick a colony from an agar-plate and inoculate LB-Media with it, incubate at 28°C/37°C overnight (ONC).
Centrifuge down the cells, remove media and resuspend in M9-minimal-media with yeast extract. Incubate overnight at 28°C/37°C.
Centrifuge down the cells, remove media and resuspend in M9-minimal-media. Incubate overnight at 28°C/37°C.

Inducing expression of fluorescence proteins for detection

Alkaline induction of alx promoter

ONC in LPM with pH 7.0
Inoculate 20 ml of pH 7.0, 8.0 and 8.5
After 20 and 40 minutes take 1 ml of each culture
Measure OD600 and fluorescence (mNeonGreen excitation 490 nm, absorbance 520 nm)
Divide fluorescence by OD600 to standardize the values

Acid induction of asr promoter

ONC in LPM with pH 7.0
Inoculate 100 ml of LPM wit pH 7.0, 5.0 and 4.5 to an OD600 of 0.2
After 1, 1.5 and 2 hours take 3 OD unit samples of each culture

Westernblot

SDS-Page

assemble electrophoresis chamber and fill it with MES buffer
load standard and samples
run SDS-page at 180V for approximately 45 min

Blotting

fill up the blotting chamber with Transferbuffer (20% Methanol) and soak sponges
assemble blotting chamber with SDS-gel, nitrocellulose membrane, filters and sponges
blot at 230 mA for 75 min

Detection

block membrane overnight with TBST milk (5%) at 4°C
wash 3 times with TBST (0,05% Tween20) for about 5 min each time
incubate membrane with peroxidase linked antibody for 2 h
wash 3 times with TBST (0,05% Tween20) for about 5 min each time
incubate with peroxidase substrate for about 1 min
detect substrate (choose detection method suitable for specific substrate)

Team:NAWI Graz/WP

Working Protocols