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.

SDS-PAGE for protein electrophoresis

Introduction

PAGE (polyacrylamide gel electrophoresis) is a technique allowing to separate charged molecule according to their molecular masses. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a variant of PAGE allowing to separate protein molecules according to their molecular masses. SDS (sodium dodecylsulphate) is a negatively charged molecule which will bind to proteins as they are heat denatured and confers them a charge nearly proportional to their length (and hence their mass). It therefore allows to separate proteins according to their molecular masses.

Materials

• Laemmli buffer (SDS-PAGE loading buffer, contains SDS and DTT)
• Commercial polyacrylamide gels in glass plates
• PAGE machine
• Running buffer (1X TGS buffer)
• Long pipet tips

Procedure

Sample preparation

1. Mix 10µl of Laemmli with 5µl of protein sample and 5µl nuclease free water in a tube. Take care to mix well the sample tube before by flicking it gently several times.
2. Incubate at 100°C for 15min in a dry heating block compatible with the tube used (e.g. for PCR tubes use the ThermoCycler). Do not forget to use lids heating at 105°C to avoid condensation.
3. The samples can now be stored at 4°C until they are loaded on the gel.

PAGE-machine preparation

4. Verify that you have all the machine's components (tank, lid with power cables, electrode assembly, cell buffer dam, casting frames and stands) as well as a new polyacrylamide gel in glass plates.
5. Rince all the components with distilled water.
6. Open the gel and remove the plastic lid that protects the bottom of the gel.
7. Open the casting frame and put the gel inside. Close it again.
8. Test waterproofness by puting new running buffer inside (here it is important to use new running buffer!). Do it above the tank (in case it is not water proof).
9. Put the casting frame in the tank. Take care of the electrodes' colors! The black wire should be above the black electrode, same for the read one.
10. Fill the part of the tank outside of the casting stand with running buffer (you can put already used running buffer here.) You should fill it until the black line drawn on the tank (the 2 gels line if you run with only 1 casting stand, the 4 gel one if you run with 2 casting stands.)
11. Close the tank with the lid with powe cables.

Loading the gel

12. Use long tips! Take care to pipet really slow in order to have the right volume. Long tips are very thin and tend not to fill completely.
13. Load 10µl of sample or 5µl of ladder per well.
14. Go to the glass plate in front of you and try to put your tip a bit in the well. Once you think you are in, try to go back and forth. If your land in the middle of the buffer, it means that you were not really in the well! You should only be able to move between the 2 glass plates in which your gel is. Once your in, dive a bit deeper in the well and load the content of your pipet.
15. Tip: put one ladder and one negative control per gel!

PAGE run

16. Set the machine on 30min and 120V. You can make sure the machine is running by looking below the gel. You should see bubbles forming.
If you do not see any bubbles, this means that the electric circuit is not closed. This could be due to the oversight to remove the plastic lid at the bottom of the gel or to the fact that there is not enough buffer in the tank or between the 2 gels. Sometimes it is necessary to put more buffer than the level indicated on the tank.
17. After 5min of run, set the voltage on 200V.

Gel wash

18. Take the casting stand out and put the now used running buffer in a bottle. You can reuse it multiple times.
19. Put the running buffer of the tank in the same bottle.
20. Remove the gel of the casting frame and open it carefully using the specialized metal instrument (or just a metallic spatule if you do not have one).
21. Put the gel with distilled water in a cylinder bowl that you can close. In order to transfer the gel from the glass plate to the bowl without breaking it, you can flip
22. Pour distilled water in order to fill the bowl up to 4cm.
23. Mix a bit and throw the water away. You can use your fingers to prevent the gel to fall in the sink.
24. Pour distilled water in the bowl and put it on the shaker for 10min.

Coomassie staining

25. Put on the shaker for 1h.
26. Wash twice with distilled water and recover the gel with distilled water. Let on the shaker overnight.

Gel visualisation

27. Your gel is now ready to be visualized. To take it out of the bowl, it is better that it is not at the bottom of the bowl. You can pour some water at one border of the bowl in order to make it float a bit and then slip your hand (with clean gloves!) underneath. Then put the gel in a plastic sleeve.
28. In order to visualize the gel, you can use a scanner (gives the best quality), or the white light mode of an UV transilluminator.

Standard heat purification for proteins

Introduction

When the protein of interest is heat stable, the heat purification method is a straightforward way to get rid of the majority of non-desired proteins of a sample.

Materials

• Protein samples to purify
• Eppendorf tubes
• Heating block compatible to the tubes used
• Ice
• Microcentrifuge

Procedure

• Denaturation
  1. Heat at 70ºC for 20 min.
  2. Put on ice for 15 min.
• Centrifugation
  • Centrifuge at 12000 g for 10 min.
  The protein of interest is now mainly located in the supernatant of the solution.