=> So we incubate all week-end at 18°C and 120 rpm.
| Composent |
Volume & Mass |
| Bactotryptone |
10 g |
| NaCl |
10 g |
| Yeast Extract |
5 g |
| H2O |
qsp 1 L |
| Adjust pH to 7.5 with NaOH and autoclave for 20 minutes |
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.
11/06/2015
Results of Transformation Efficiency of chemical competent cells v2
Low efficiency because we regenerate on LB (without glucose) normally, it is either SOC or LB + glc.
Petri dish too small is boring
All small colonies (strange ???) => it was bubbles => is boring
Preparation Glucose 2M stock (150mL)
Dissolve 50,48 g glucose into 150 ml (final volume) ddH2O and filter-sterilize into sterile 500 ml tube. Store at 4°C.
- Transformation Chemical cells (Azote) with pSB3C3, pSB3T5, (+pSB1C3 control)
Materials :
Resuspended DNA (Resuspend well in 10µl dH20, pipette up and down several times, let sit for a 5 minutes)
Competent cells (50µl per transformation)
Ice (in ice bucket/container)
2ml tube (1 per a transformation')
42ºC water bath
LB media (10 mL LB + 100µL glucose 2M)
Petri dishes with LB agar and appropriate antibiotic (2 per transformation)
Petri dishes with LB agar without antibiotic (1 per transformation)
glass spreader
37ºC incubator
10pg/µL RFP Control (pSB1C3 w/ BBa_J04450)
Protocol
0. Resuspended well in 10µl dH20, pipette up and down several times, let sit for a 5 minutes
Spin down the DNA tubes 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.
Thaw competent cells on ice. Label one 2.0mL microcentrifuge tube for each concentration and then pre-chill by placing the tubes on ice.
Pipet 1 µL of DNA into each microcentrifuge tube (pSB3C3, pSB3T5, pSB1C3). For each concentration, use a separate tube.
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.
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.
Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
Add 200 µL of LB+glucose (20mM final) media per tube, and incubate at 37°C for 2 hours.
Prepare the agar plates during this time: label them,
Label two petri dishes with LB agar and the appropriate antibiotic (ChL+Tet) with the part number, plasmid backbone, and antibiotic resistance.
Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
For the control, label two petri dishes with LB agar (ChL). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
For control bacterial survey, plate 20µL on one LB agar without antibiotic by transformation
Incubate at 37°C overnight. Position the plates so the agar side is facing up, and the lid is facing down.
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.
=> OBSERVATION the 15/06
11/06/2015
Medium Preparation
- LB agar (X2) 500ml = 20 Petri dishes 10cm
Pouring the Plates
1. Make sure bench top has wiped down with bleach/EtOH.
2. Remove sterile Petri dishes from plastic bag
3. Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not
lift the cover off excessively (you should be able to just open up enough to pour).
4. Swirl plate in a circular motion to distribute agar on bottom completely.
5. Let each plate cool until its solid (~20 minutes) then flip so as to avoid
condensation on the agar.
6. Store plates in plastic bags in 4°C room with: name, date and contents
M63 Medium
- 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.