1. Pellet 1–5 ml (not to exceed 15 OD units) bacterial culture by centrifugation for 30 seconds. Discard supernatant. 1.5 ml of culture is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) . Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Add 200 μl Plasmid Lysis Buffer (B2) , gently invert tube 5–6 times, and incubate at room temperature for 1 minute. Color should change to dark pink, and solution will become transparent and viscous. Do not vortex.
4. Add 400 μl of Plasmid Neutralization Buffer (B3) , gently invert tube until neutralized, and incubate at room temperature for 2 minutes. Sample is neutralized when color is uniformly yellow and precipitate forms. Do not vortex.
5. Centrifuge lysate for 2–5 minutes. For best results, and especially for culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Pellet should be compact; spin longer if needed. continued on back → For a detailed protocol or to download the full manual, visit www.neb.com/T1010. Monarch® Plasmid Miniprep Kit Protocol Card
6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Centrifuge for 1 minute. Discarding the flow-through is optional.
8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute.
10. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. Nucleasefree water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA, (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.

1. Prepare a liquid culture of the bacteria by incubating a bacterial sample in 5-10 ml LB Medium overnight
2. Dilute Glycerol with distilled water to creat a 50% Glycerol solution
3. Transfer 750 ul of the glycerol solution into a microfuge tube
4. Add 750 ul of bacterial culture to microfuge tube
5. Close lid and mix gently
6. Freeze at –80 C

1 L 50x TAE Buffer:

1. Tris base 2M (242 g)
2. Acetic acid 1M (57 ml)
3. EDTA 0.05 M (100 ml 0.5 M)
4. Fill up to 1 L with MilliQ H2O

Dilute 50x TAE to 1x TAE for use.

1. Cast 50 ml 1% agarose gel: (1g agarose, fill up to 100 ml with 1x TAE buffer)
2. Load gel, including at least one lane for a DNA ladder
3. Run gel at 120V for 45 min

Gel extraction using QIAgen DNA Gel extraction kit, https://www.qiagen.com/us/resources/resourcedetail?id=a72e2c07-7816-436f-b920-98a0ede5159a&lang=en

PCR mix:

Component	Volume	Final Conc
5X Q5 Reaction Buffer (NEB)	10 µl	1X
dNTP Mix, 10 mM each	1 µl	0.2 mM each dNTP
Fwd primer	2.5 µl	0.5 µM
Rev primer	2.5 µl	0.5 µM
Q5® High-Fidelity DNA Polymerase (NEB)	0.5 µl	0,02 u
DNA to be amplified	1 µl	< 1,000 ng
ddH2O	32.5 µl	32.5 µl
Total	50 µl	-

PCR program:

Step	Temp/°C	Duration/sec	n repeats
Denaturation	98	30	1
Denaturation	98	10	30
Annealing	depends on primers	30	30
Extension	72	120	30
Extension	72	120	1
Cooling	7	120	1

Q5 reaction mix

Component	Volume	Final Conc
5X Q5 Reaction Buffer (NEB)	10 µl	1X
dNTP Mix, 10 mM each	1 µl	0.2 mM each dNTP
Fwd primer	2.5 µl	0.5 µM
Rev primer	2.5 µl	0.5 µM
Q5® High-Fidelity DNA Polymerase (NEB)	0.5 µl	0,02 u
ddH2O	33.5 µl	33.5 µl
Total	50 µl	-

1. Mix reaction mix for each colony to be analyzed
2. Pick colony using disposable pipette tip
3. Stab pipette tip onto replica plate and note number
4. Mix pipette tip in PCR mix
5. Run PCR (program see below)
6. Analyze PCR using agarose gel to test if insert of expected size was amplified
7. Incubate replica plate overnight

PCR program:

Step	Temp/°C	Duration/sec	n repeats
Denaturation	94	300	1
Denaturation	94	60	34
Annealing	depends on primers	30	34
Extension	72	60	34
Extension	72	60	1
Cooling	7	120	1

1. Inoculate liquid overnight cultures in 500ml LB Medium (+Amp), for the negative control Kanamycin was added instead of Ampicillin.
2. Let grow at 37°C and shaking until an OD600 of about 0,6 is reached (2-3 h)
3. Take a 1 ml Sample before induction
4. Induce expression with 1mM IPTG
5. Let Cultures incubate at 25 °C overnight
6. Cells were centrifuged for 20 min at 4 °C, supernatant was discarded afterwards
7. resuspend in a 50mM Tris/HCl, 500 mM NaCl (ph 8) Buffer A with DNase and Protein inhibitor
8. Cells were lysed using a Sonicator for 12 min each
9. Lysate was centrifuged at 4°C for 20 min
10. Lysate was filtered using a Syringe

Purification with Ni Column

1. Add 10 ml ddH20 to column
2. Add 10 ml Wash Buffer (Buffer A+ 20 mM Imidazol)
3. Add 25 ml Celllysate, collect 1 ml Flowthrough for analysis
4. Add 15 ml Wash Buffer
5. 14 x 1 ml Elutionbuffer (Buffer A + 500 mM Imidazol), catch elute in 1.5 ml reaction tubes
6. Add 15 ml Wash Buffer
7. To rejuvinate Column:
8. Add 15 ml 50 mM EDTA
9. Add 15 ml Wash Buffer
10. Add 15ml 0.1M NiSo4
11. Add 10 ml wash buffer
12. Add 15ml 20% EtOH

1. Add ≤80µl of Enzyme Reaction Mix per well to a white opaque 96-well plate. Note: Less than 80µl may be applied to accommodate an added volume of test compounds (e.g., 70µl enzyme reaction mix plus 10µl of test compound).
2. Add test compounds (such as drugs or other small molecules or vehicle) to controls without a test compound (e.g., DMSO in same concentration as present in wells with test compounds).
3. Incubate enzyme reaction at desired temperature (e.g., 37°C or room temperature) for the desired length of time. Note: Various methods of initiating and terminating enzyme reactions may be employed. For example, reactions may be initiated by addition of an essential reaction component and stopped by addition of an enzyme inhibitor. The final volume of the reaction should be 80µl to allow for addition of H2 O2 Substrate solution.
4. After the enzyme reaction incubation, add 20µl of H2 O2 Substrate solution and mix.
5. Incubate the reaction for 60 minutes at room temperature (approximately 22°C).
6. Add 100µl ROS-Glo™ Detection Solution
7. Incubate 20 minutes at room temperature.
8. Read relative luminescent values (RLU) using a plate reading luminometer.

1. Prepare dilution sequences of the Protein Fraktion and one control group with BSA BSA Dilutionsequence in NaCl, Tris Puffer: 1 mg/ml, 0,5 mg/ml, 0,25 mg/ml, 0,125 mg/ml, 0,0625 mg/ml, 0 mg/ml Fraktion Dilutionsequence: 1:1 diluted, 1:5 diluted, 1:10 diluted, 1:100 diluted
2. Add 196 μl of Bradford Reagent and 4 μl of Sample to a well Plate
3. Measure with a spectrometer and use control as reference

Materials

pre-made 10% SDS gel

Protein Marker

1x SDS runnning buffer

electrophoresis chamber

Coomassie Staining colution

Procedure

1. add buffer to sample
2. heatshock at 95°C for 10 min
3. load gel with samples and protein marker and run at 150 V for 60 min
4. rinse gel with VE water, then incubated with Coomassie Staining colution while shaking
5. rinse the gel twice with VE water