Difference between revisions of "Team:UPMC-Paris/Experiments"

Competency and transformation of E. coli

Preparation of E. coli competent cells :

E. coli Calcium Chloride competent cell protocol

1.Inoculate a single colony into 5mL Lb in 50mL falcon tube. Grow O/N @ 37°C.
2.Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.
3.Shake @ 37°C for 1.5-3hrs.

Or

1.Inoculate a single colony into 25mL LB in a 250 mL bottle in the morning.
2.Shake @ 37°C for 4-6 hrs.

Then....

1.Put the cells on ice for 10 mins (keep cold form now on).
2.Collect the cells by centrifugation in the big centrifugue for 3 mins @6krpm
3.Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
4.Incubate on ice x 20 mins
5.Centrifuge as in 2
6.Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol
7.Dispense in microtubes (300μL/tube). Freeze in -80°C.

Transformation of E. coli competent cells :

1.Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2.Remove agar plates (containing the appropriate antibiotic ) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3.Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube.
GENTLY mix by flicking the bottom of the tube with your finger a few times.

Note: Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid.

4.Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5.Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).
6.Put the tubes back on ice for 2 min.
7.Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.

Note: This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded on the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.

8.Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.

Note: We recommend that you plate 50 μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

Note: If the culture volume is too big, gently collect the cells by centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before the cells begin dividing, the bacteria diffuse through the liquid and won't grow in colonies.

9.Incubate plates at 37°C overnight.

Competency and transformation of B. subtilis.

Preparation of B. subtilis competent cells

Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C
The following morning scrape the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium (20 ml) to give an OD600 reading of about 0.5.
Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.
When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 min) inoculate 200 ml of pre-warmed, SpII medium with 2 ml of stationary-phase culture and continue incubation at 37°C with slower aeration
After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.
Carefully decant the supernatant into a sterile container and save.
Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently
Aliquot the competent cell (0.5 ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-iced/ethanol bath or ice/isopropanol bath and store -70°C.

Transformation of B. subtilis competent cells

Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath
Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently
In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.
Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.

Vector modifications

Digestion :

Select restriction enzymes to digest your DNA. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
In a 1.5mL tube combine the following:

1. ➟ DNA
2. ➟ Restriction Enzyme(s)
3. ➟ Buffer
4. ➟ dH2O up to total volume

Mix gently by pipetting.
Incubate tube at appropriate temperature (usually 37°C) for 1 hour.
Always follow the manufacturer’s instructions.
To visualize the results of your digest, conduct gel electrophoresis

Vector Preparation :

Combine the following in a PCR or Eppendorf tube:

1. ➟ Vector DNA
2. ➟ Insert DNA
3. ➟ Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)
4. ➟ 0.5-1μL T4 DNA Ligase
5. ➟ H20 to a total of 10μL

To dertermine the amount of DNA needed, we used the NEB calculator
Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).

Other

Gel Extraction:
We followed the protocol provided in the kit, you can dowload it here
We resuspended DNA in 50 uL water.

Plasmid Extraction:
We followed the quick protocol provided in the kit, you can see it here
We resuspended DNA in 30 - 50 uL of DNase free water.

PCR Purification:
We followed the quick protocol provided in the kit, you can download it here
We resuspended our purified PCR products in 50 uL water.

PCR:
We followed the protocol provided by the manufacturer, you can see it here
We use different cycles programs depending on what we wanted. You can see our Notebook for more informations.

Dephosphorylation of 5' -ends:
Using Alkaline Phosphatase we use the following protocol:

1) Make a mix of 1 pmol of DNA (about 1 ug of 3kb plasmid), 2 uL of Phosphatase Buffer, 5 units of Phosphatase and water to reach 20 uL

2) Incubats 30 minutes, 37°C

Heat inactivation at 80°C for 20 minutes

Ligation:
Using T4 DNA Ligase we perform this protocol:
Reaction setup

Total reaction volume can be adjusted as needed.

1. ➟ Add all of above components to a clean reaction vessel, mix well by pipetting.
2. ➟ Incubate at 25 °C for 30 minutes.
3. ➟ Immediately purify DNA using PCR clean-up column and elute in ~50 µL.
4. ➟ OR - Immediately dilute (at least 1:10, but enough such that 0.1-10 ng of ligation product will be transformed in TE or water
5. ➟ Transform 0.1-10 ng of ligation product into chemically or electrocompetent cell line that is compatible with vector