Notebook

Day 1

Description of everything we do

02/06/2015

- Stock solution MgSO4 : 0,5M

Pour 500mL :

61,62g de MgSO4 (MM : 246,48)

qsp 500mL H2O stérile

- SOB Medium : 1 L

- 2XTY Medium 500 mL

03/06/2015

- Transformation Broth (TB): 1 L

Mix the Pipes, CaCl2, and KCl in 900 ml of millipore water. Add NaOH until pH is 6.7,

Don't worry, dust disappear after pH adjust.

Add MnCl2 (see below), stir, adjust volume to 1 L, then filter sterilize. Store at 4C.

- Making DMSO Competent Cells : DAY ONE

Grow 12 ml overnight culture of favorite strain of E. coli in 2XTY at 37°C and 250 rpm (We use Denis’ DH5-alpha).

04/06/2015

- Making DMSO Competent Cells : DAY TWO

Inoculate 1 L SOB with 12 ml overnight culture.

Keep 5mL of SOB for initial OD.

Grow culture at 18C and 180 rpm (this temperature is really important as we see a 10-fold decrease in competency when we grow them at room temperature).

- Solution stock Chloramphenicol 34 mg.mL-1 (29,5 mL) :

Weigh out 1 g chloramphenicol and dissolve in 25 ml of 100% ethanol.

Make up volume to 29.5 ml with 100% ethanol.

Sterilize by filtration.

STORAGE : Aliquots of appropriate volume can be stored at -20 °C

- Solution stock Tetracycline 12,5 mg.mL-1 (80 mL) :

Weigh out 1 g tetracycline and dissolve in 75 ml of 1:1 vol/vol distilled water:ethanol.

Make up volume to 80 ml with 1:1 vol/vol distilled water:ethanol. (40mL ethanol + 40mL dH2O)

Sterilize by filtration.

STORAGE : Aliquots of appropriate volume should be wrapped in aluminium foil and stored at -20 °C

05/06/2015

-LB agar 250mL (x3)

-LB liquid 500mL

- Making DMSO Competent Cells : DAY THREE

Grow cells until A600 0.5-0.7

Blanck = SOB

A600 nm = 0,011

A600 nm = 0,008

=> So we incubate all week-end at 18°C and 120 rpm.

08/06/2015

- Making DMSO Competent Cells : DAY SIX

Grow cells until A600 0.5-0.7

Blanck = SOB

A600 nm = 0,008

=> So we take an other protocol (openwarewet)

-LB liquid 1L (x3)

5X stock of M63 Medium (1L)

- Add the following reagents to a 2-liter flask:

- 10 g (NH4)2SO4

- 68 g KH2PO4

- 2.5 mg FeSO4.7H2O

1 liter of high quality distilled water

Once the ingredients are added, heat with stirring until the components are completely dissolved.

Adjust to pH 7 with acid.

Autoclave at 121°C for 20 min.

PROBLEMS : we have FeSO4 precipitate + pH superior to 7

Hypothesis : we had put 2,5g of FeSO4.7H2O instead of 2,5mg

Preparation of other « Making Chemical Competent cells » DAY ONE

TSS buffer

To make 50 mL:

5g PEG 8000 (Denis Friend)

1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)

Add LB to 50 mL

Filter sterilize (0.22 μm filter)

Add after sterilization 2.5 mL DMSO (the 09/06)

Overnight culture

Grow a 5mL overnight culture of DH5-alpha in LB media at 37°C and 180 rpm.

09/06/2015

Preparation of other « Making Chemical Competent cells » DAY TWO

1. In the morning, dilute this culture back into 50mL of fresh LB media in a 200mL conical flask, at 37°C and 180 rpm. (Dilute the overnight culture by at least 1/100).

Grow the diluted culture to an OD600 of 0.2 - 0.5.

We obtained : OD600nm = 2,744

SO to obtain a good quantity of cells. We « dilute » culture later

2. Put eppendorf tubes on ice now.

- Theoretically, if your culture is 50 ml, you will need 50 tubes. But we had 5X more cells. We will need 250 tubes.

- At this point you should also make sure that your TSS is being chilled (it stored at 4°C).

3. Split the culture into two 50mL falcon tubes and incubate on ice for 10 min.

TARE : tube 1 = 37,03g

tube 2 = 37,00g

All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible

4. Centrifuge for 10 minutes at 3000 rpm and 4°C.

5. Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.

6. Resuspend in chilled TSS buffer.

The volume of TSS to use is 10% of the culture volume that you spun down => so 25 mL

You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall. We resuspend with

pipetteman with a cone cut at its end.

7. Add 100 μl aliquots to your chilled eppendorfs and store at − 80°C.

One part was stored directly at -80°C and the other part was freeze in liquid nitrogen.

10/06/2015

- Transformation Efficiency Kit iGEM

Protocol

1. Spin down the DNA tubes from the Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. Note: There should be 50 µL of DNA in each tube sent in the Kit.

2. Thaw competent cells on ice. Label one 2.0mL microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.

3. Pipet 1 µL of DNA into each microcentrifuge tube. For each concentration, use a separate tube.

4. Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C.

5. Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

6. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.

7. Add 200 µL of LB media per tube, and incubate at 37°C for 2 hours.

8. Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.

9. Pipet 20 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Do triplicates (3 each) of each tube if possible

10. Incubate at 37°C overnight. Position the plates so the agar side is facing up, and the lid is facing down.

11. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.