=> 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
12/06/2015
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.
15/06/2015
Ampicillin stock solution (50 mg ml-1)(40 ml)
Weigh out 2 g ampicillin (sodium salt) and dissolve in 35 ml of distilled water.
Make up volume to 40 ml with distilled water.
Sterilize by filtration.
Aliquots of appropriate volume should be stored at -20 °C.
For culture plates, allow media to cool to 55 °C before adding ampicillin to a final concentration of 50 μg/ml.
Kanamycin stock solution (50 mg ml-1)(40 ml)
Weigh out 2 g kanamycin sulfate and dissolve in 35 ml of distilled water.
Make up volume to 40 ml with distilled water.
Sterilise by filtration.
Aliquots of appropriate volume can be stored at -20 °C.
For culture plates, allow media to cool to 55 °C before adding kanamycin to a final concentration of 50 μg/ml.
Counting of Transformation Chemical cells (Azote) with pSB3C3, pSB3T5, (+pSB1C3 control)
- Reception of the Nicolas’ Request for yeast plasmid to iGEM HQ
Bba_J63005 ,Amp-R : orange cap
Bba_K319043, Chlor-R : green cap
Select Clones pSB3T5 and pSB3C5
Picking Colonies Protocol
After taking the plates out of the 37ºC incubator place them upside down (i.e. the way they were in the incubator) on the bench top.
Using a pipette boy or similar instrument, pipette 5 ml of LB media*** (containing the correct concentration of antibiotic into sterile 25 ml or 50 ml tubes
(The number of tubes depends on how many you want to grow)
Tetracyclin : 12,5 µg/mL
Chloramphenicol : 10 µg/mL
In one hand take a sterile pipette tip on the end of a pipette, with the other hand pick up the upside down plate containing the bacteria from the ligation. Turn the plate over in your hand so that the bacteria are now facing upwards towards you and touch the tip of the pipette tip gently to a bacterial colony that is completely isolated from any other colony.
=> The Petri dishes are stock in parafilm at 4°C.
Now place the same tip with bacteria on it into one of the tubes containing LB media and move the tip around a bit to release some of the bacteria into the liquid. Some people simply eject the pipette tip into the media but if you do this you will need to recover it the next day.
Culture the tubes overnight in an incubated orbital shaker at 37ºC at 190-225 rpm.
*** Preparation of LB media :
Chloramphenicol : 5mL x 12 colony + 5 = 65mL
Chloramphenicol (final) : 10 µg/mL
Chloramphenicol (stock) : 34 mg/mL
Tetracyclin : 5mL x 1 colony + 5 = 10 mL
Tetracyclin (final) : 12,5 µg/mL
Tetracyclin (stock) : 12,5 mg/mL
Vi = (Cf x Vf) / Ci
20µL of Chloramphenicol into 65 mL LB media
10µL of Tetracyclin into 10 mL LB media
=> 12 colony for pSB3C5
=> 1 colony for pSB5T5
Next Morning :
For pSB3C5 : Colony 1,2,3,4,5,6 and 11 = Red pellet / Colony 7,8,9,10 and 12 = No pellet
For pSB3T5 : Colony 1 = Red pellet
16/06/2015
Medium Preparation
- LB agar 250ml (x2) = 10 Petri dishes 10cm
Pour 20 Petri dishes.
Store at 4°C.
- Preparation of Nicolas’ Request yeast plasmid
Bba_J63005 ,Amp-R : orange cap
Bba_K319043, Chlor-R : green cap
The agar will have a hole from when it was stabbed.
Dip an inoculating loop into the stab
Plate onto a petri dish of LB agar with Ampicillin or Chloramphenicol
Incubate the dish overnight at 37C (14-16hr)
- Storage of pSB1AK3 (plate 4, well 12B) and psB1C3+Bba_I14033 (plate3-13D)
- Resuspended well in 10µl dH20, pipette up and down several times, let sit for a 5 minutes
- Transfert into eppendorf tube
- Storage 4°C
Transformation with pSB1AK3 (plaque 4, well 12B), psB1C3+Bba_I14033 (pCAT) and psB1C3 temoin
Note : For Temoin, 100µL bacteria and 45sec at 42°C => For testing
- Miniprep on selected colony for pSB3C5 and pSB3T5
Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge on Select Clones pSB3T5 and pSB3C5
This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium.
Note: All protocol steps should be carried out at room temperature.
=> Tube 1,2,3,4,5,6 and 10 for pSB3C5
=> Tube 1 for pSB3T5
Procedure
0. Centrifuge culture 6min at 4400g.
1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a
microcentrifuge tube.
2. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix.
Do not vortex
Do not allow the lysis reaction to proceed for more than 5 min.
3. Add 350 µl Buffer N3 and invert the tube IMMEDIATELY but GENTLY 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.This step is required for low copy number plasmid.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
To elute DNA,add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep
spin column, let stand for 1 min, and centrifuge for 1 min.
11. Storage at 4°C
17/06/2015
- Verification of Purification of Plasmid by Restriction digest (NotI)
8 Digest (7 pSB3C5 + 1 pSB3T5)
1. Quickly vortex all ingredients before beginning.
2. Add the following in a micro-centrifuge tube:
5µL NEBuffer 3
3µL ADN (normally 1µg)
41µL dH2O
1µL NotI enzyme (NEB for tube 1,1’ and Fermentas for tube 2,3,4,5,6,10)
3. Incubate reaction in a 37°C water bath for at least one hour.
Agarose gel 2% : 1g agarose + 50 mL TBE
70mV, DNA ladder 100kb and 1 kB.
Select Clones pSB3T5 and pSB3C5
Picking Colonies Protocol
After taking the plates out of the 37ºC incubator place them upside down (i.e. the way they were in the incubator) on the bench top.
Using a pipette boy or similar instrument, pipette 5 ml of LB media*** (containing the correct concentration of antibiotic into sterile 25 ml or 50 ml tubes
(The number of tubes depends on how many you want to grow)
Ampicillin+Kanamycin : 12,5 µg/mL
Chloramphenicol : 10 µg/mL
In one hand take a sterile pipette tip on the end of a pipette, with the other hand pick up the upside down plate containing the bacteria from the ligation.
Turn the plate over inyour hand so that the bacteria are now facing upwards towards you and touch the tip of the pipette tip gently to a bacterial colony that is completely isolated from any other colony.
=> The Petri dishes are stock in parafilm at 4°C.
Now place the same tip with bacteria on it into one of the tubes containing LB media and move the tip around a bit to release some of the bacteria into the liquid. Some people simply eject the pipette tip into the media but if you do this you will need to recover it the next day.
Culture the tubes overnight in an incubated orbital shaker at 37ºC at 190-225 rpm.
*** Preparation of LB media :
Chloramphenicol : 5mL x 12 colony = 70mL
Chloramphenicol (final) : 10 µg/mL
Chloramphenicol (stock) : 34 mg/mL
Ampicillin+Kanamycin : 5mL x 2 colony = 10 mL
Ampicillin+Kanamycin (final) : 50 µg/mL
Ampicillin+Kanamycin (stock) : 50 mg/mL
Vi = (Cf x Vf) / Ci
20µL of Chloramphenicol into 70 mL LB media
10µL of Ampicillin + 10µL of Kanamycin into 10 mL LB media
=> 10 colony for pSB1C3-Bba_I14033 (pCat- + 2 colony for Bba_K31943 (iGEM request)
=> 4 colony for pSB1AK3 (Nicolas backbone)
pSB3C5-Bba_J04450 = 3807pb => NotI (1079 and 3793)
pSB3T5-Bba_J04450 = 4321pb => NotI (1079 and 4307)
Bba_J04450 = 1069pb
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