Team:UPNAvarra Spain/Protocols

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Protocols

Index

1. Competent cells 2. Transformation via electroporation 3. Streak plate method 4. Glycerol stocks 5. E.coli liquid culture 6. Plasmid DNA isolation 7. PCR 8. Electrophoresis 9. Biobrick assembly

1. Competent cells

We generated electrocompetent E. coli Top10 cells.

Materials:

LB-Medium
Streptomycin
Autoclaved 10% glycerol (cooled)
Autoclaved 125ml centrifuge tubes
Ice bucket and ice
Centrifuge

Method:

Inoculate 25 mL LB-Strep with bacterial stock, incubate over night at 37 °C, 200 rpm.
Inoculate 1 L LB-Strep with 20 ml of the over night culture and incubate at 37 °C, 200 rpm until OD₆₀₀ = 0.6.
Store on ice for 15 minutes.
Divide the cultures into the pre-cooled 250 ml centrifuge tubes and centrifuge at 4000 g, 4 °C, 15 min with slow acceleration and decceleration.
Discard supernatant and resuspend the pellets in 125 ml cooled 10 % glycerol shaking gently.
Centrifuge at 4000 g, 4°C, 15 minutes.
Discard supernatant and resuspend the pellets in 125 ml cooled 10 % glycerol shaking gently. Join the resuspensions in two tubes.
Centrifuge at 4000 g, 4 °C, 15 minutes.
Discard supernatant and resuspend in 20 ml cooled 10 % glycerol shaking gently.
Centrifuge at 4000 g, 4 °C, 15 minutes.
Discard supernatant and add a final volume of 2.5 ml cooled 10 % glycerol and resuspend shaking gently.
Aliquot in 50 µL and freeze in liquid nitrogen immediately.
Store at -80 °C.

2. Transformation via electroporation

Materials:

Plasmid DNA
Electrocompetent cells (E.coli Top10)
Ice bucket and ice
1 mm path electrocuvettes
1.5 ml microcentrifuge tubes
An electroporator
LB medium
LB agar plates with appropriate antibiotic

Method:

Thaw 50 µL electrocompetent E. coli cells on ice.
Add 1 µL resuspended DNA (kit) or 2 µL ligation to 50 µL electrocompetent cells.
Electroporate at U = 1.25 kV, C = 25 µF, R = 200 Ω.
Transfer transformation reaction to 1 ml LB medium and shake 1 h at 37 °C, 200 rpm.
Plate on selective LB medium.
Incubate over night at 37 °C .

3. Streak plate method

Materials:

Bacterial culture
Sterile petri dish with selective LB medium
Inoculating loop
Bunsen burner

Method:

TSterilize the wire loop.
Cool the loop by touching it on the edge of the sterile agar plate.
Dip the loop into the broth bacteria culture.
Drag the loop over the surface of the top one-third of the plate back and forth in a "zig-zag" formation.
Sterilize the loop in the flame.
Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three times and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate without touching that area again.
Sterilize the loop in the flame.
Turn the plate 90 degrees. Repeat the procedure above.
Incubate the plate for 24 hours at 37°C.

4. Glycerol stocks

Materials:

LB medium
Microentrifuge tubes
50% Sterile glycerol
Liquid nitrogen

Method:

Pick a colony, inoculate LB medium with the appropiate antibiotic and grow overnight at 37°C, 200 rpm.
Add 0.5 ml of the culture to 0.5 ml of 50 % sterile glycerol in a microcentrifuge tube.
Vortex and freeze the glycerol on liquid nitrogen.
Store at -80°C.

5. E.coli liquid culture

Materials:

LB Medium
Sterile glass tube
Antibiotics

Method:

Pipette 3 mL LB medium into a sterile glass tube.
Add the corresponding antibiotic.
Inoculate the tuve with a single colony from the plate.
Leave it shaking at 37 ℃ overnight.

6. Plasmid DNA isolation

Materials:

1.5 ml microcentrifuge tubes
Solutions I, II and III
Ice bucket and ice
Centrifuge

Solution I: 50 mM Glucose, 10 mM EDTA and 25 mM Tris-HCl, pH 8.0 (+ RNAse 1mg /ml). Solution II: 0.2 N NaOH, 1% SDS.
Solution III: 3 M Potassium acetate, 2 M Glacial acetic acid.
Ethanol: 100 % and 70 % (cooled).

Method:

Thaw RNAsa and prepare solution I, II and III.
Fill the eppendorf with bacterial culture, centrifuge (14000 rpm, 3 min) and remove the supernatant.
Add 175 µl of solution I + RNAsa (fresh) and resuspend the pellet.
Add 175 µl of solution II and mix by inversion, incubate 5-10 min on ice.
Add 175 µl of solution III and mix by inversion, incubate 5-10 min on ice.
Centrifuge at 14000 rpm, 5 min at 4°C.
Transfer the supernatants to new microcentrifuge tubes.
Add 850 µl of cooled 100 % ethanol and mix by inversion.
Incubate 20 min at -20°C.
Centrifuge at 14000 rpm, 5 min at 4°C.
Discard the supernantant and add 500 µl of cooled 70 % ethanol.
Centrifuge at 14000 rpm, 5 min at 4°C.
Discard the supernatant and let the pellet dry at room temperature.
Add 20 µl H20 mQ and resuspend the pellet pipetting.
Store at -20°C.

7. PCR

A) Phusion PCR (Thermo Fisher)

Phusion master mix (50 µL):

10 µL 5 x HF-buffer
1 µL 10 mM dNTPs
1 µL template (plasmid 1:100 dilution)
0.5 µL Phusion DNA polymerase
1 µL forward primer 25 µM
1 µL reverse primer 25 µM
35.5 µL dH2O

Phusion standard protocol:

Initial denaturation: 95°C – 5 minutes.
30 cycles of:
- Denaturation: 95 °C - 45 seconds.
- Annealing: T (°C) - 45 seconds.
- Elongation: 72 °C - 30 s/kb.
Final elongation: 72 °C - 10 minutes.
Storage: 4 °C.

Gel electrophoresis and band purification:

B) Taq polymerase (Biotools)

Master mix (25 µL):

3 µL 10x buffer
1 µL 10 mM dNTPs
1 µL template (plasmid 1:100 dilution)
1 µL DNA polymerase
1 µL forward primer 10 µM
1 µL reverse primer 10 µM
17 µL dH2O

Standard protocol:

Initial denaturation: 95°C – 5 minutes.
30 cycles of:
- Denaturation: 95 °C - 45 seconds.
- Annealing: T (°C) - 45 seconds.
- Elongation: 72 °C - 60 s/kb.
Template alternative
- Pick colony, streak at marked position on a new plate and solute remaining cells on the tip in the PCR tube with reaction mixture, cultivate if insert is of correct size.
Final elongation: 72 °C - 10 minutes.
Storage: 4 °C.

Gel electrophoresis for control of fragment size:

8. Electrophoresis

Materials:

1x TAE buffer
DNA ladder
Power supply
DNA samples
Agarose
Casting tray
Gel box
Midori green

50x TAE buffer: 2 M Tris, 1 M Glacial acetic acid, 50 mM EDTA disodium salt.

Method:

Weight agarose and puti t in a Erlenmeyer flask.
Add the requiered volumen of 1x TAE and swirl the content.
Microwave the agarose until melt completely.
Prepare a casting tray while the agarose solution cools down.
Add midori green, swirl, poor the solution in the casting tray, insert a comb and let it solidify.
Refill the gel box with 1x TAE buffer and put the casting tray in the gel box.
Load the DNA ladder.
Add 1/6 volume of loading buffer to the DNA simples and load them in the gel.
Run the gel at 90 V until the dye line is approximately 75-80% of the way down the gel.
Turn off power, disconnect the electrodes from the power source, and then remove the gel from the gel box.
Using any device that has UV light, visualize the DNA fragments.

9. Biobrick assembly

A) Digestion

Materials:

Part samples (miniprepped)
Plasmid backbone (miniprepped)
Restriction enzymes (EcoRI, XbaI, SpeI, PstI) and buffers (NEB)
BSA
dH20
Heat block

Method:

Digestion for plasmid backbone (20µl total):
- 2 µl NEB buffer 3.
- 0.2 µl BSA.
- 1 µl EcoRI.
- 1 µl XbaI.
- 2 µl Plasmid.
- 13.8 µl dH20.
Digestion for the part (20 µl total):
- 2 µl NEB buffer 2.
- 0.2 µl BSA.
- 1 µl EcoRI.
- 1 µl SpeI.
- 2 µl Part.
- 13.8 µl dH20.
Incubate at 37°C in a heat block for 1 hour.
Inactivate the enzymes for 15 min at 65°C.

B) Desphosphorylation

Materials:

Plasmid digestion
Alkaline phosphatase CIAP (Roche) and buffer
Heat block

Method:

Add 1 µl of CIAP (1U/µl) to 20 µl digestion.
Incubate for 30 min at 37 °C.
Inactivate the enzyme 15 min at 65 °C.

C) Electrophoresis

D) DNA purification from gel

Materials:

Ultra Clean 15 DNA Purification kit (Mobio)
1.5 ml microcentrifuge tubes
Razor Blade
Heat block
Centrifuge

Method:

Excise gel slice containing the DNA fragment using a clean razor blade and place it into a 1.5 mL tube.
Record the weight of the gel slice and add 450 µL Ultra Salt solution to 0.1 g gel slice.
Incubate the mixture at 55 °C until the gel slice is completely dissolved.
Add 6 µl of matrix, mix by inversión and incubate 5 min at room temperature.
Centrifuge at 14000 rpm, 45 s and discard the supernatant.
Resuspend the pellet in 1 ml Ultra Wash solution.
Centrifuge at 14000 rpm, 45 s and discard the supernatant x 2.
Resuspend the pellet in 12 µl os dH2O and incubate 5 min at room temperature.
Centrifuge at 14000 rpm, 75 s and transfer the supernatant to a new microcentrifuge tube.
Store at -20 °C.

E) Ligation

Materials:

Linearized and purified plasmid backbone
Digested part
T4 DNA ligase (Invitrogen) and buffer

Method:

Add 1 µl of digested plasmid backbone.
Add 4 µl of digested part.
Add 4 µl T4 DNA ligase buffer.
Add 1.5 µl T4 DNA ligase.
Add water to 20 µl.
Ligate 1 hour at room temperature.

F) Transformation via electroporation

G) Colony PCR