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Protocols

Protocols

Growth media

LB medium

Material

2 g Tryptone

1 g Yeast extract

1 g NaCl

200 ml dH₂O

Method

Using a 1L conical flask add 200ml dH2O.

Add 2 g Tryptone, 1 g yeast extract, 1 g NaCl to 200ml dH2O, mix until dissolved.

Close with sponge and foil.

Autoclave 125 ^oC/15 min

Store at room temperature.

LB agar

Material

500 mg Tryptone

250 mg Yeast extract

250 mg NaCl

750 mg Agar

dH₂O to 50 ml

Method

2 x 50 ml in 125 ml flask

Close with sponge and foil.

Autoclave 125 ^oC/15 min

store at room temperature.

SOC media

For 1 x 10ml solution, stored in 20ml culture tubes with a screw cap add the following:

0.2 g Bacto Tryptone

0.05 g Bacto Yeast Extract

20 µl of 5M NaCl

25 µl of 1M KCl

100 µl of 1M MgCl₂

100 µl of 1M MgSO₄

200 µl of 1M glucose

Adjust to 10 ml with dH₂O

Sterilize by autoclaving and store at room temperature.

0.1M MgCl₂ solution

2.033 g of MgCl₂ ·6H₂O in 100 ml of H₂O in 100 ml Pyrex bottle.

Autoclave and store at 4 ^oC.

0.1M CaCl₂ solution

11 g of CaCl₂ ·6H₂O in 500 ml of H₂O in 500 ml Pyrex bottle.

Autoclave and store at 4 ^oC.

50% glycerol solution

5 ml of glycerol and 5 ml H₂O in 20 ml culture tube with screw cup.

Autoclave and store at 4 ^oC.

Antibiotic stock solutions

Ampicillin 100 mg/ml – 1 g in 10 ml of MilliQ water

Kanamycin 15 mg/ml – 0.15g in 10 ml of MilliQ water

Tetracycline 12.5 mg/ml – 0.125 g in 10 ml of 50% v/v ethanol

Chloramphenicol 34 mg/ml – 0.34 g in 10 ml of 80% v/v ethanol

The stock solutions is filtered through a 0.2 m m filter, aliquoted and stored at -20^oC until use. The final working concentration of all antibiotics is 1:1000 of the stock solution.

Making competent cells

Preparing cells

1. Work under flame.

2. Streak 10 ml of E. coli cells onto one of the LB agar plates containing no antibiotic or specific for the cell type.

3. Grow cell overnight at 37 ^oC (plate upside down), no more than 16 h.

4. Wrap plate in a parafilm and store at 4 ^oC.

   Making competent cells

1. Pick single colony of the cells from the LB agar plate into 10 ml of LB media containing no antibiotic or specific for the cell type. Grow the cultures overnight at 37 ^oC with shaking at 250 rpm.

2. Inoculate 200 ml of prewarmed to 37^oC LB medium (no antibiotics or specific for the cell type) with 10 ml of the overnight cultures, and grow at 37^oC for 60 min, with vigorous shaking 250 rpm or until the OD₆₀₀ is 0.4 - 0.5.

3. Put flask on ice for 30 min. At the same time chill sterile falcon (centrifuge) tubes.

4. Aliquot culture into 50 ml each 4 x 50 ml chilled falcon (centrifuge) tubes.

5. Harvest the cells by centrifugation for 7 min at 3500 rpm, at 4 ^oC and discard supernatants completely.

6. Resuspend cells in each tube in 12.5 ml of 0.1 M MgCl₂.

7. Centrifuge for 7 min at 3500 rpm, at 4^oC and discard supernatants.

8. Resuspend cells in each tube in 25 ml of 0.1 M CaCl₂.

9. Incubate cells on ice for 30 min.

10. Centrifuge for 7 min at 3500 rpm, at 4^oC and discard supernatants.

11. Resuspend cells in each tube in 700 ml of 0.1 M CaCl₂ and 300 ml of 50% glycerol. Final volume 1 ml in each tube.

12. Aliquot 50 ml aliquots into 1.5 ml sterile microcentrifuge tube on ice and

    store at –80^oC.

    Bacterial Transformation

1. Take 50 µl of prepared E. coli competent cells and put on ice for 5 min

2. Add 1µl of plasmid DNA and incubate on ice for 5 min

3. Heat-shock for 1 min at 42 ^oC

4. Put on ice for 5min

5. Add 250 µl of room temperature SOC buffer

6. Incubate for 1 h at 37 ^oC with shaking at 250 rpm

7. Plate on one LB agar Petri dish with appropriate antibiotic

8. Incubate at 37 ^oC overnight

9. The next day count cell colony, wrap plate in a parafilm and store at 4 ^oC.

   Spin column plasmid purification protocol

   1. Resuspend cell pellets from 5 ml of overnight culture in 250 ml of Buffer D1 and transfer into 1.5 ml microcentrifuge tube.

   2. Add 250 ml of Buffer D2 and mix.

   3. Add 350 ml of Buffer N3 and mix.

   4. Centrifuge sample at 13,000 rpm for 10 min (at room temperature).

   5. Collect supernatant.

   6. Place a spin column in a provided 2 ml collection tube.

   7. To bind DNA, apply the sample to the column.

   8. Centrifuge for 1 min. Discard flow-through and place the column back into the same tube.

   9. To wash, add 500 ml Buffer DB to the column.

   10. Centrifuge for 1 min. Discard flow-through and place the column back into the same tube.

   11. To wash, add 750 ml Buffer DE to the column.

   12. Centrifuge for 1 min. Discard flow-through.

   13. Place the column back into the same tube.

   14. Centrifuge the column for an additional 1 min and place column in a clean 1.5 ml microcentrifuge tube.

   15. To elute DNA, add 30 ml of H₂O, let the column stand for 1 min.

   16. Centrifuge the column for 1 min.

   17. Store purified DNA samples at -20 ^oC

   Gel Electrophoresis

   Stock solutions

   5x TAE buffer

   24.2 g Tris base (200mM)

   5.7ml Glacial acetic acid (100mM)

   3.72g Na₂EDTA (5mM)

   Dissolve and mix into 1L dH₂O

   Autoclave

   Store at room temperature

   Agarose Gel

For ?% agarose gel	Agarose (g)	Agarose (g)
0.8%	0.24	0.48
1.0%	0.30	0.60
1.5%	0.45	0.90
2.0%	0.60	1.20
Final volume	30ml	60ml

1. Into a 125 ml Erlenmeyer borosilicate glass flask weigh out and dissolve required amount of agarose in either 30ml or 60ml 1x TAE buffer (for DNA samples less than 20µl use 30ml of agarose. For DNA samples of 30-50µl use 60ml agarose).

2. Heat in microwave until dissolved (approximately 1 min at 700 w)

3. Cool solution to 50 ^oC.

4. Add 1µl Gel Red stain to flask

5. Pour the gel into the prepared 7cm x 7cm gel tray with 1.5mm fixed height comb.

6. Allow to set for 15-30m mins

7. Remove any tape from the gel mold and place the gel mold in the running tank with the DNA samples at the cathode (black) end.

8. Fill the running tank with 250ml 1x TAE buffer

9. Mix 10µl of DNA sample with 2µl of 6x sample buffer.

10. Load each sample into the wells of the gel .

11. Run at 100V for 45-60mins

Gel analysis

If your DNA samples are intended for cloning purposes, use a long wavelength UV light (366nm) gel viewer. Use a clean scalpel to cut the gel with the bands present and store in a 1.5ml microcentrifuge tube for further extraction and purification.

If there is no need to use the DNA for cloning purposes use the standard short wavelength UV light (254nm) viewer.

Spin column DNA extraction from the gel protocol

1. Excise the DNA fragments from the gel with a clean, sharp scalpel. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer DG to a 1 volume of gel.

2. Incubate sample at 50 ­­⁰C for 10 min, mix by vortexing every 2-3 min.

3. Add 1 gel volume of isopropanol to the sample and mix.

4. Place a spin column in a provided 2 ml collection tube

5. To bind DNA, apply the sample to the column.

6. Centrifuge for 1 min, discard flow-through and place the column back into the same tube

7. (Optional) Add 0.5 ml of Buffer DG to column and centrifuge for 1 min

8. Centrifuge the column for 1 min, discard flow-through and place the column back into the same tube

9. To wash, add 0.75 ml Buffer DE to the column

10. Centrifuge for 1 min and discard flow-through.

11. Place the column back in the same tube.

12. Centrifuge the column for an additional 1 min and place column in a clean 1.5 ml microcentrifuge tube

13. To elute DNA, add 30 ul of H₂O and let the column stand for 1 min.

14. Centrifuge the column for 1 min

15. Store purified DNA samples at -20 ^oC.

Digestion

30 µl of plasmid

4 µl of 10x Cutsmart buffer

1.0 µl of Restriction enzyme

1.0 µl of Second restriction enzyme

1 µl dH₂O

Incubate tube at 37 ^oC for 60 minutes.

Ligation

7µl of gene

1 µl of plasmid

1 µl of 10x DNA ligase buffer

1 µl of 10x DNA ligase

Incubate at room temperature for 60 mins

PCR

Working solutions

10µl of forward primer + 90µl of RNase-free water

10µl of reverse primer + 90µl of RNase-free water

PCR mixture

In your strip PCR tubes add the following:

Master Mix 5µl

Forward primer 2.5µl

Reverse primer 2.5µl

Sample DNA 1µl

PCR water 14µl

Control tube:

Master Mix 5µl

Forward primer 2.5µl

Reverse primer 2.5µl

PCR water 15µl

PCR Machine Cycling Times and Temperatures:

Step		Temperature(oC)	Time
Initial Denaturation		95	30 sec
24 Cycles	Denaturation	95	30 sec
	Annealing	52	30 sec
	Extension	72	30 sec
Final Extension		74	5-10 min

Serial dilution of IPTG: IPTG was provided in a stock solution of

1. Set up 22 UBs and label

2. Add 5mL of LB broth

3. Add 5 mL chloramphenicol

4. Inoculate appropriate bottles (all but 1 and 2)

5. Add IPTG to 2, 4, 5, 6, 7

6. Place all UBs in shaking incubator at 37 degrees.

7. When 0.4 < OD600 < 0.6, add IPTG to bottles 8, 9, 10, 11

8. Incubate overnight

9. The following day, pipet UB contents into 96-microtiter well plate

10. Measure absorption with machine of 7th floor

	Tube number	Description
Controls	1	LB broth
	2	No cells, 1mM IPTG (50mL)
	3	Cells, no IPTG
IPTG at T0	4	Cells, 1 mM IPTG (50mL)
	5	Cells, 100 mM IPTG (50mL)
	6	Cells, 10 mM IPTG (50mL)
	7	Cells, 1 mM IPTG (50mL)
IPTG at 0.4 < OD600 < 0.6	8	Cells, 1 mM IPTG (50mL)
	9	Cells, 100 mM IPTG (50mL)
	10	Cells, 10 mM IPTG (50mL)
	11	Cells, 1 mM IPTG (50mL)

Performed for Top10, DH5alpha

Biofilms

8 plates were made:

#1. Negative control using broth of PSB1C3

#2. Broth of PpuI

#3. AHL with water

#4. AHL with DMSO

#5. Rsal 2

#6. Rsal 4

#7. LapG

#8. DMSO

1. Inoculate putida KT2440 into 3-5mL of LB

2. Incubate overnight in shaking incubator at 37 C

3. Inoculate 1mL of putida into 100mL of LB (1:100 dilution)

Serial Dilutions

Negative control / PpuI / Rsal 2/ Rsal 4/ LapG

stock

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

10-9

Stock contains: 1000 microL

Other 9 tubes contain 900 microL LB and 100 microL of previous tube

DMSO

stock

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

10-9

Stock contains 1000 microL of DMSO

Other 9 tubes contain 900 microL of water + 100 microL of previous tube

AHL with water:

stock

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

Stock contains diluted AHL into DMSO

Other 9 tubes contain 900 microL of water + 100 microL of previous tube

AHL with DMSO:

stock

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

Stock contains diluted AHL into DMSO

Other 9 tubes contain 900 microL of DMSO + 100 microL of previous tube

LB	Putida	10-9	10-8	10-7	10-6	10-5	10-4	10-3	10-2	10-1	stock
LB	Putida	10-9	10-8	10-7	10-6	10-5	10-4	10-3	10-2	10-1	stock
LB	Putida	10-9	10-8	10-7	10-6	10-5	10-4	10-3	10-2	10-1	stock
LB	Putida	10-9	10-8	10-7	10-6	10-5	10-4	10-3	10-2	10-1	stock

Unless for AHL which did not have 10-9, so column 3 starts at 10-8.

Setting up the plates:

4. Pipette 200 microL of LB into column 1 (rows A-D) in all 8 plates

5. Pipette 200 microL of diluted putida into column 2 (rows A-D) in all 8 plates

6. Pipette 100microL of diluted putida into columns 3-12 (rows A-D) in all 8 plates