Protocols

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

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%).

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.