(Difference between revisions)
 Revision as of 15:54, 5 June 2012 (view source)m ← Previous diff Revision as of 16:51, 5 June 2012 (view source)m Next diff → Line 221: Line 221: ===Day 3=== ===Day 3=== - Note the time (for later reference) + TIme: Warm up nutrition: Warm up nutrition: Line 243: Line 243: *Sample: 100µl of culture + 900µl of nutrition *Sample: 100µl of culture + 900µl of nutrition *At OD600=1.5, stop growing and start pelleting of cell *At OD600=1.5, stop growing and start pelleting of cell - ---- - Note the time + Time: - Remember: keep bottles and tubes cool at all steps except 80min pre-incubation + ''Remember: keep bottles and tubes cool at all steps except 80min pre-incubation'' Transfer bacteria culture to 500mL centrifuge bottles: Transfer bacteria culture to 500mL centrifuge bottles: Line 270: Line 269: *parameters: rotor number: 20, speed: 4000rpm, time: 8min, temperature: 4°C *parameters: rotor number: 20, speed: 4000rpm, time: 8min, temperature: 4°C Discard supernatant and centrifuge it again for 2 min, and then remove the residue of supernatant by pipetting Discard supernatant and centrifuge it again for 2 min, and then remove the residue of supernatant by pipetting - ---- - Note the time + Time: - • Weigh the mass of 50mL of centrifuge bottle (with pellet) + - unit Bottle 1 Bottle 2 Bottle 3 Bottle 4 + Weigh the mass of 50mL of centrifuge bottle (with pellet) + {| border="1" + |- + !  !!  unit  !!  Bottle 1  !!  Bottle 2  !!  Bottle 3  !!  Bottle 4 + |- + | Empty bottle || mg || || || || + |- + | Bottle with pellet || mg || || || || + |- + | Mass of cell || g || || || || + |- + | Volume of S30 buffer A || mL || || || || + |- + | Mass of bead (mass of cell x 5.0) || g || || || || + |- + | Mass of bead (mass of cell x 0.05)        || g || || || || + |- + | Mass of bead || g || || || || + |} + unit Bottle 1 Bottle 2 Bottle 3 Bottle 4 Empty bottle mg Empty bottle mg Bottle with Pellet mg Bottle with Pellet mg

## Protocol for E. Coli Crude Extract Preparation, 3X

Time needed: 3 days

### Materials

2xYT:

• 2xYT-broth powder (MP biomedicals)
• Cat #: 3012-032

Bacto-Agar:

• Bacto-agar (BD)
• Product number: 214010

• 0.1mm diameter glass beads (Biospec products)
• Catalog #: 11079101

• Catalog #: 3110BX

• Polypropylene microvials (Biospec products)
• Catalog #: 522S

• Micro bio-spin chromatography columns (Bio-Rad)
• Product number: 732-6204

Centrifuge (big size):

• Allegra 64R high-speed refrigerated benchtop centrifuge (Beckman Coulter)

Centrifuge (ultra size):

• Beckman J2-M1 (Beckman Coulter)

Centrifuge bottle (50ml):

• Polyallomer bottle with screw-on cap (Beckman Coulter)
• Part number: 357003

Centrifuge bottle (500ml):

• Centrifuge bottle assembly polycarbonate (Beckman Coulter)
• Part number: 355607

Chloramphenicol:

• Chloramphenicol powder (Sigma-Aldrich)
• Product number: C1919

Cuvette:

• Fisherbrand Disposable Cuvettes
• Cat#: 14-955-127

Dialysis cassette:

• Slide-A-Lyzer Dialysis Cassette, 10MWCO (Thermo Scientific), maximum capacity: 3mL
• Product number: 66380

E. coli strain:

• BL21 Rosetta 2 competent cell (Novagen)
• Product number: 71402

Electrode:

• Electrode, 3-in-1 for Φ 400, 500 series pH meter, 12x 150 mm, Epoxy
• Product number: A57198

• Product number: 26500 (4000ml)

Floater:

• Float Buoys: comes with Slide-A-Lyzer Dialysis cassette

Incubator shaker:

• Incubator shaker (New Brunswick)
• Innova 4300

K-glutamate:

• L-Glutamic acid potassium salt monohydrate (Sigma Aldrich)
• Product number: G1149

Liquid nitrogen

Magnetic stirrer:

• Thermolyne Nuova II
• Fisher Scienetific Isotemp Digital Magnetic Stirrer

Mg-glutamate:

• L-Glutamic acid Hemimagnesium salt tetrahydrate (Sigma Aldrich)
• Product number: 49605

PH meter:

• pH meter, Φ 410 pH/mV meter, waterproof, handheld
• Product number: A58734

Potassium phosphate monobasic solution:

• Potassium phosphate monobasic solution (Sigma Aldrich)
• Product number: P8709

Potassium phosphate dibasic solution:

• Potassium phosphate dibasic solution (Sigma Aldrich)
• Product number: P8584

Protein assay:

• Product number: 500-0006

Spectrometer:

• Genesys 10 uv (Thermo Electron corporation)

Table top centrifuge:

• Microcentrifuge model 5415D (Eppendorf)

Table top vortex:

• Analog vortex mixer (VWR)
• Catalog number: 58816-121

Tris:

• Tris base, white crystals or crystalline powder, molecular biology (Fisher Scientific)
• Catalog number: BP 1 52 1

### Ingredients

2xYT broth:

• Concentration of 2xYT: 31g/L
• Dissolve 62g of 2xYT powder with 1.8L of water in total volume → the rest of volume will be filled by phosphate and water later

2xYT + Phosphate + Chloramphenicol plate: (2xYT+P.P.+Ch.)

• Concentration of 2x YT: 31g/L
• Concentration of chloramphenicol: 34mg/ml
• Concentration of phosphate: 62ml of phosphate/ 1L of total volume
• Weigh 1.24g of 2x YT, 0.44g of Bacto-Agar
• Transfer 2xYT and Bactor-Agar in a bottle, add water up to 40ml and then autoclave it
• When temperature is about 50°C after autoclaving, add 2.5ml of phosphate and 60μl of chloramphenicol at 34mg/ml (proper quantity is 40μl, but I add 50% more because it is still hot (~50°C))
• Mix well and pure 25ml of mixture on petri dish (100X15mm)
• Cure it on room temperature for 1 hour

Chloramphenicol:

• Concentration of chloramphenicol stock: 34mg/ml
• FW: 323.13
• Dissolve 6.8mg of chloramphenicol powder with 200μl of alcohol in total volume

Phosphate:

• 100ml of potassium phosphate dibasic solution + 55ml of potassium phosphate monobasic solution

S30 buffer A (2 liters) :

• Final concentration and pH: 14mM of Mg-glutamate, 60mM of K-glutamate, pH7.7
• Weigh 10.88g of Mg-glutamate and 24.4g of K-glutamate
• Transfer powders into 2L beaker with magnetic stirrer
• Add water up to 1.9L and place it on magnetic stir plate
• Run plate, start to measure pH of solution and keep running plate and measuring pH
• Add 50mM of Tris: 50ml of Tris at 2M
• Add Acetic Acid up to reach ph7.7
• Add water to fill up to 2L
• Add DTT to 2mM final concentration just before use.

S30 buffer B (2 liters):

• Final concentration and pH: 14mM of Mg-glutamate, 60mM of K-glutamate, pH8.2
• Weigh 10.88g of Mg-glutamate and 24.4g of K-glutamate
• Transfer powders into 2L beaker with magnetic stirrer
• Add water up to 1.9L and place it on magnetic stir plate
• Run plate, start to measure pH of solution and keep running plate and measuring pH
• Add Tris up to reach pH8.2
• Add water to fill up to 2L
• Add DTT to 1mM final concentration just before use.

Tris:

• Concentration of Tris: 2M
• FW: 121.14
• Dissolve 60.57g of powder with 250ml of water in total volume

### Pre-Preparation

Clean all materials:

• 4L Erlenmeyer glass flask (erlens)
• 500ml centrifuge bottles
• 50ml centrifuge bottles
• Funnel
• Beaker
• Magnetic stirrer
• Floater
• Rinse all with dH2O
• Rinse floater and magnetic stirrer with isopropanol
• Burn at 110°C except floater

### Day 1: Transformation

Start to prepare 2xYT+P.P.+Ch. plate at around noon (follow the ingredients protocol)

At around 5pm, start transformation:

• Take E. coli strain stock (33% glycerol) out from -80°C
• Pick some of stock and dissolve it into 500ml of 2xYT broth (Immediately place back E. coli strain stock into -80°C)
• Do plating with 5~10µl of dissolved E. coli stock and place it in 37°C incubator
• Check DTT at -40°C: it is aliquot by 1ml at 1M.
• 6 tubes will be used: 4 for S30 buffer A and 2 for S30 buffer B

### Day 2: Miniculture

Start the 1st miniculture at noon:

• Start to warming up at 11:30
• 4ml of 2xYT, 0.27ml of phosphate and 4µl of chloramphenicol
• Pick one colony from plate and start miniculture at noon

Prepare 2xYT, phosphate and S30 buffer A and B (follow the ingredients protocol)

Start the 2nd miniculture at 8:30:

• Start to warming up at 8:00
• 50ml of 2x YT, 3.3ml of phosphates, 50μl of chloramphenicol and 100μl of miniculture

When warming up at 8:00, prepare other materials for tomorrow morning:

• 6 of 4L erlens: wrap their opening up with aluminum foil and place them on bench
• 6 of rubber caps: wrap them up with aluminum foil and place them on bench
• 2 of 1.8L 2xYT bottles: place them on bench
• Phosphate bottle: place it on bench
• Water (autoclaved): place it on bench
• 4 of 500ml centrifuge bottles: place them on bench
• 4 of 50ml centrifuge bottles: place them on bench
• Funnel: wrap it with aluminum foil and place it on bench
• S30 buffer A and B bottles: place them in chromato
• Glass bead bottle: put it into 110°C oven
• Two rotors with lids for J2-M1: place them in chromato

### Day 3

TIme:

Warm up nutrition:

• Add 124ml of phosphate into 1.8mL of 2xYT and add water to fill up to 2L → 2 bottles
• Transfer 660mL of nutrition into 4L erlen → 6 erlens
• Incubator shaker at 220rpm at 37°C

Induce 6.6ml of the 2nd culture (last night) into each 4L erlen 30min after warm-up

• It takes about 3 h ~ 3 h 20 min

Meanwhile, check other materials:

• Take glass bead bottle out from the oven
• Place a rotor for 500mL centrifuge bottle onto J2-M1 and turn the machine on
• parameters: rotor number: 10, speed: 5000rpm, time: 12min, temperature: 4°C
• Place a rotor for 15mL falcon tube onto Allegra64R and turn the machine on
• parameters: rotor number: F0850, speed: 7500rpm, time: 10min, temperature: 2°C
• Turn on the spectrometer
• Weigh 50ml centrifigue bottles: look at the table below
• Add 4mL of DTT at 1M into S30 buffer A

Check OD after 3 hours incubation of bacteria culture:

• Set the wavelength at 600nm
• Control: 1mL of nutrition
• Sample: 100µl of culture + 900µl of nutrition
• At OD600=1.5, stop growing and start pelleting of cell

Time:

Remember: keep bottles and tubes cool at all steps except 80min pre-incubation

Transfer bacteria culture to 500mL centrifuge bottles:

• Use 4 bottles, and all must be balanced to prevent damaging J2-M1, which is very powerful

Centrifuge it

• parameters: rotor number: 10, speed: 5000rpm, time: 12min, temperature: 4°C

Discard supernatant and repeat the previous step

Discard supernatant and add 200mL of S30 buffer A, and then resuspend the pellet cells in the bottle:

• make sure to shake vigorously by hand until no cell clusters remain

Centrifuge it with the same parameters and repeat the previous step

Prepare 37.5mL of S30 buffer A in 4 x 50mL falcon tubes

Discard supernatant and pour 37.5mL of S30 buffer A (prepared the previous step)

Resuspend the pellet cell gently (vortex by hand)

• it doesn’t need to be resuspended completely

Transfer pellet cell into 50mL centrifuge bottle through funnel and centrifuge it (change rotor)

• parameters: rotor number: 20, speed: 4000rpm, time: 8min, temperature: 4°C

Discard supernatant and centrifuge it again for 2 min, and then remove the residue of supernatant by pipetting

Time:

Weigh the mass of 50mL of centrifuge bottle (with pellet)

unit Bottle 1 Bottle 2 Bottle 3 Bottle 4
Empty bottle mg
Bottle with pellet mg
Mass of cell g
Volume of S30 buffer A mL
Mass of bead (mass of cell x 5.0) g
Mass of bead (mass of cell x 0.05) g

```    Fill them with mixture and spin quickly to remove air in the tubes
Add little bit of mixture on top of mixture in the tubes up to slight below of tube height
Add also little bit of mixture inside on cap
Close tubes tightly
```