Protocols

This page collects the different protocols used in our project. They are sorted in alphabetical order.

Competent cell transformation (with Amplicilin)

Introduction

This protocol shows how to transfer plasmid DNA into competent cells.

Materials

• Competent cells
• Control plasmid
• ligation mix
• LB-Ampicilin plates
• Heating Block
• Bunsen burner
• Ethanol 96%

Procedure

1. Add components according to the following table to three tubes of competent cells

Amounts in μl	Transfection mix	Vector control
Competent cells (In tube)	50μl	50μl
Plasmid DNA	5μl	-
Vector	-	5μl

2. Incubate on ice for 30 min.
3. Heat shock the cells up to 45 sec. at 42°C. Immediatly transfer the tube back on ice for 5 min.
4. Spread 50μl.
5. Incubate the plates overnight at 37°C to select for transformants.

DPNI plasmid digestion

Introduction

DpnI cleaves only when it's recognition site is methylated. Useful for removing cell-derived plasmid template from PCR samples.

Materials

• DPNI
• Enzyme buffer (Might work with the one used for the PCR)
• PCR product

Procedure

Digest mix

PCR product	50μl
DPNI	1μl

Incubation

Incubate for one hour at 37°C

DPNI heat inactivation

incubate at 80°C for 20 minutes

Inoculating cultures

Introduction

This protocol explains how to inoculate cultures to grow bacterial clones.

Materials

• LB ampicillin plates from our transformation
• LB ampicillin medium
• 14ml sterile round tubes with dual position snap cap
• sterile tips
• shaker at 37°C

Procedure

1. Pick a colony from the ligation plate using a sterile tip
2. Shake the tip into a bacterial culture tube containing 3ml of LB/Amp medium so the colony mixes with the medium
3. Close tubes (loose position for sterile aerobic culturing)
4. Put your tubes onto a shaker at 37°C and incubate overnight with agitation at 225 rpm

Glycerol stock preparation

Introduction

This is how to make glycerol stocks of bacteria cell cultures that are suitable for long time storage

Materials

• Liquid cell culture
• Glycerol
• 1.5 ml tube

Procedure

1. After you have bacteria growth in your liquid culture, add 500μl of overnight culture to 500μl of 50% glycerol in the 1.5ml tube and gently mix
2. Freeze the glycerol stock tube at -80°C. The stock is now available for years as long as its kept at -80°C.
3. To remove bacteria from the glycerol stock, open the tube and use a sterile tip to scrape some of the frozen bacteria.

Oligomer Phosphorylation

Introduction

This protocol is used to phosphorylate the 5' ends of inserts used in a subsequent Golden Gate ligation reaction

Materials

• Forward Oligo 100 μM
• Reverse Oligo 100 μM
• T4 DNA Ligase Buffer 10X
• PNK
• NFW
• NaCl 2M aqueous solution

Procedure

1. In a PCR tube mix the following (total volume 29 μL):
• 3 μL Forward Oligo 100 μM
• 3 μL Reverse Oligo 100 μM
• 3 μL T4 DNA Ligase Buffer 10X
• 2 μL PNK
• 18 μL water
2. Incubate the mixture for 2 hours at 37C
3. Heat inactivate PNK at 65C for 20 minutes
4. Add 1 μL of 2 M NaCl aqueous solution
5. Heat to 98C for 2 minutes then slowly ramp down to room temperature and hold at 4C when finished

Preparation of dumbbell probes

Introduction

The goal is to prepare dumbbell probes in order to amplify miRNAs by Rolling Circle Amplification (RCA).

Materials

• 2 μL DNA template
• 1μL T4 polynucleotide kinase
• 1 μL ( 100 U/μL ) T4 ligase
• 4 μL T4 DNA ligase reaction buffer (x10) (2μL for the phosphorylation and 2μL for the ligation)
  • 400 mM Tris-HCl, 100 mM MgCl2, 100 mM Dithiothreitol, 5 mM ATP, pH 7.8 at 25 °C
• 22 μL DEPC-treated H2O (15 μL for phosphorylation and 7 for ligation)
• Exonuclease I (20 U/μL) and Exonuclease III (100 U/μL)

Procedure

Phosphorylation of the oligos

1. In the IDT tubes, put the amount of water to get 100μM of DNA probe [you take the number of moles N and suspend in a volume of 10*N μl]
2. In a tube, put 2 μL oligos, 15 μL of water, the T4 ligase buffer (2 μl), and finally 1 μL of the kinase.
3. Incubate the mixture at 37°C for 1 hour.
4. Heat at 60°C for 20 min to inactivate the enzyme.
   

   The buffer has to be new ( less than 1 year) and we should avoid repeated freeze-thaw cycle with it.

Ligation of the probes

5. Add in a reaction tube 10 μl (because at the end of phoshorylation is 10 μM and not 100 μM) of DNA template, the T4 ligase (1 μl), the reaction buffer (2 μl) and 7 μl DEPC-treated water.
6. Put the tube at 16°C for 2 hours to process the ligation.
7. Then heat at 65°C for 10 min to terminate the reaction.
8. Add the exonucleases (1μl each - total volume of 22 μl) and incubate the reaction mixture at 37°C for 2 hours.
9. Then the enzymes are denatured by heating at 80°C for 20 min.
10. The ligation can be controlled by electrophoresis on agarose gel (1.5%).

Real-time fluorescence measurement of RCA

Introduction

The goal is to make sure that the RCA worked fine for different concentration of probes and miRNAs. The initial assessement was done with 0.5% agarose gel but the results were difficult to interpret. We are using 25x SYBR Green I, an intercalating agent for dsDNAs that is also fluorescent (Excitation wavelength is 494 nm, emission wavelength is 521nm).

Materials

• 1 μL prepared probes
• 2.5μL phi29 DNA polymerase reaction buffer (x10)

500 mM Tris-HCl, pH 7.5 at 25°C, 100 mM MgCl2, 100 mM (NH4)2SO4, 40 mM Dithiothreitol

• 0.25 μL BSA (20 mg/mL)
• 6 μL dNTPs(10 mM for each)
• 2.5 μL of target miRNA solution
• 11.75 μL DEPC-treated H2O
• 0.5 μL phi29 DNA polymerase (10 U/μL)
• 0.5 μl 25x SYBR Green I

Procedure

Fluorescence measurement using SYBR Green I

1. The SYBR Green I that we purchased is optimised for Gel and so it was very concentrated (x10000) so we have to dilute it to 25x with the polymerase buffer.
2. Put all the components in a tube
3. Inject 24 (25) μl in a 96-wells plate and put it in the plate reader.
4. The reaction should be at 37°C. The florescence is measured every 2 min during 180 min under excitation and emission wavelengths of 495(497) and 515(520) nm, respectively.

Rolling Circle Amplification

Introduction

The goal here is to amplify miRNAs by RCA (Rolling circle amplification). The dumbbell probes are designed in order to get a complementary region with specific miRNAs. The miRNAs bind to this region and the probes become circular and the amplification can begin. We finally obtain a concatemer (long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series).

Materials

• 1 μL prepared probes
• 2.5μL phi29 DNA polymerase reaction buffer (x10)
• 500 mM Tris-HCl, pH 7.5 at 25°C, 100 mM MgCl2, 100 mM (NH4)2SO4, 40 mM Dithiothreitol
• 0.25 μL BSA (20 mg/mL)
• 6 μL dNTPs(10 mM for each)
• 2.5 μL of target miRNA solution
• 12.25 μL DEPC-treated H2O
• 0.5 μL phi29 DNA polymerase (10 U/μL)
• 2 μL SYBR ISYBR I (x10)

Procedure

Amplification of the miRNAs

1. Add all the component, except the last one in a 25μL mixture tube.
2. Incubate the mixture at 37°C for 2h
3. Heat it at 65°C for 10 min to stop the reaction.
4. The mixture can be analysed by using electrophoresis or fluorescence analysis.

Fluorescence analysis

5. mix the mixture with 2 μL SYBR I (x10).
6. load into a 37 °C pre-warmed 384-well plate.
7. The florescence is measured every 2 min during 180 min under excitation and emission wavelengths of 495 and 515 nm, respectively.