Team:Patras/Protocols
Protocols
- Genomic DNA extraction from whole blood samples with DNA extraction Kit (based on the kit user manual and adjusted on the BentoLab device specifications)
Before starting the preparation:
- Prepare Washer Buffer B5 by adding 48 mL ethanol 96-100% as indicated on the label. After the addition mark the label of the bottle that the ethanol was added.
- Prepare the lyophilized Proteinase K by adding the indicated volume of Proteinase4 Buffer PB to dissolve it.
- Incubate samples at 70 °C for 10–15 min.
- Preheat Elution Buffer BE to 70 °C.
1) Lyse blood sample Pipette
- 25 μL Proteinase K and up to 200 μL blood, body fluid sample, or buffy coat from 1 mL blood (equilibrated to room temperature) into 1.5 mL microcentrifuge tubes (not provided).
- Add 200 μL Buffer B3 to the samples and vortex the mixture vigorously (10–20 s).
- Set an incubator or water bath to 70 °C.
- Preheat Elution Buffer BE to 70 °C.
2) Adjust DNA binding conditions
- Add 210 μL ethanol (96–100 %) to each sample and vortex again.
3) Bind DNA
- For each preparation, take one NucleoSpin® Blood Column placed in a Collection Tube and load the sample. Centrifuge 1 min at 7.000 x g. If the samples are not drawn through the matrix completely, repeat the centrifugation at higher g-force. Discard Collection Tube with flow-through.
4) Wash silica membrane
- 1st wash: Place the NucleoSpin® Blood Column into a new Collection Tube (2 mL) and add 500 μL Buffer BW. Centrifuge 1 min at 7.000 x g. Discard Collection Tube with flow-through. + 500 μL BW 7.000 x g 1 min.
- 2nd wash: Place the NucleoSpin® Blood Column into a new Collection Tube (2 mL) and add 600 μL Buffer B5. Centrifuge 1 min at 7.000 x g. Discard flow-through and reuse Collection Tube.
5) Dry silica membrane
- Place the NucleoSpin® Blood Column back into the Collection Tube and centrifuge 1 min at 7.000 x g.
6) Elute highly pure DNA
- Place the NucleoSpin® Blood Column in a 1.5 mL microcentrifuge tube (not provided) and add 100 μL preheated Buffer BE (70 °C). Dispense buffer directly onto the silica membrane. Incubate at room temperature for 1 min. Centrifuge 1 min at 7.000 x g.
- Nanodrop
- Raise the sampling arm and wipe both the upper and lower pedestals with which the sample will be in contact, using a dry, lint-free laboratory wipe.
- Open the desired PC application.
- Pipette 1 μl of the blanking buffer BE that was used on the last step of the extraction of the DNA to elute the DNA and lower the sampling arm.
- Select the "Blank" command using the software on the PC to measure and store the reference spectrum.
- Wipe the blank from both measurement pedestal surfaces with a dry laboratory wipe.
- Raise the sampling arm and pipette 1 μl of the sample onto the lower measurement pedestal.
- Lower the sampling arm and select the "Measure" command using the software on the PC.
- When the measurement is complete, raise the sampling arm and wipe the sample again from both the upper and lower pedestals.
- PCR Allele-Specific
Initially, it should be highlighted that in PCR protocol it is recommended to use high fidelity Taq DNA Polymerase because of its 3’ to 5’ exonuclease activity. On the contrary, in AS-PCR this kind of Taq Polymerase could potentially remove the mismatch bases that are essential for AS-PCR, so it is suggested to use only Taq polymerase which lacks 3’ to 5’ exonuclease activity.
Amplification:
Calculate the appropriate concentration of PCR reagents by multiplying the given concentrations for one sample, on the following table with A (A= the number of samples + 1 blank sample + 1 extra).
| PCR reagents | Initial concentrations | Adding volume for one (1) sample | Adding volume for N samples |
|---|---|---|---|
| Distilled H2O (ddH2O) | 17 μl | 17 * A μl | |
| Buffer Solution | 10x | 2.5 μl | 2.5 * A μl |
| MgCl2 | 25 mM | 2.5 μl | 2.5 * A μl |
| Forward primer for the ancestral allele (Fw) / Forward primer for the alternative allele (Fm) | 100 u/μl | 0.75 μl | 0.75 * A μl |
| Reverse primer | 100 u/μl | 0.75 μl | 0.75 * A μl |
| dNTPs | 100 u/μl | 0.25 μl | 0.25 * A μl |
| Taq Polymerase | 5 u/μl | 0.25 μl | 0.25 * A μl |
| DNA template | 50 - 100 ng/μl | The volume of DNA template is depended on the initial concentration of the sample |
The Master mix consists of all the above PCR reagents , homogenized, added from the higher to the lower volumes. Two master mixes are prepared with a different combination of primers. The one contains the forward primer for the ancestral allele and the common reverse primer (Fw + R). The other one, contains the forward primer for the alternative allele and the common reverse primer (Fm + R). We divide each master mix into PCR tubes (24 μl) and each of the DNA samples is loaded in 2 PCR tubes (1 μl). Also, there are 2 blank samples that contain ddH2O instead of DNA sample, in the same volume (1 μl). Place the PCR tubes into the PCR device in BentoLab and adjust the proper PCR conditions and PCR cycles at 30.
| Steps | Temperature | Time |
|---|---|---|
| 1 Initial denaturation | 95℃ | 5 min |
| 2 Denaturation | 95℃ | 30 sec |
| 3 Annealing | 65℃ | 50 sec |
| 4 Elongation | 72℃ | 120 sec |
| - Repeat steps 2-4 for 30 cycles - | ||
| 5 Final elongation | 72℃ | 10 min |
| 6 Holding | 15℃ | infinity |
- Electrophoresis
Prepare a 1% agarose gel dissolved in TBE 1x at high temperature in a microwave oven. When agarose has been dissolved, add 7 μl of gel stain. Adjust the gel on the electrophoresis device of the Bento Lab, create wells and leave it for at least 30 min.
We note that better visualized results are obtained using the blue LED transilluminator and not in a UV device while using the gel stain.
- TBE recipe
TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA.
In molecular biology, TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary cofactors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg2+ is also a cofactor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low (typically at around 1 mM).
More recently discovered substitutes for TBE and TAE buffers for electrophoresis are available.
10x TBE (1 L)
- Dissolve 108 g Tris and 55 g Boric acid in 900 ml distilled water.
- Add 40 ml 0.5 M Na2EDTA (pH 8.0) (Alternative use 9.3 g Na2EDTA)
- Adjust volume to 1 L.
- Store at room temperature.
Note: 10x TBE may take some time to dissolve, even with fast stirring
10x TBE (1 L)
- Dissolve 10.8 g Tris and 5.5 g Boric acid in 900 ml distilled water.
- Add 4 ml 0.5 M Na2EDTA (pH 8.0) (Alternative use 9.3 g Na2EDTA)
- Adjust volume to 1 L.
- Store at room temperature.
Note: 0.5x TBE may also be used for electrophoresis.
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16 students from Patras blending Pharmacogenomics with Artificial Intelligence to redefine medicine
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