S2 File The Protocols of the laboratory practical lessons

In a graduated cylinder prepare 1L of solution diluting stock solution; with deionized water: 20mL of TAE 50x and 980mL of deionized water are required

Prepare the tray to add agarose solution, adding two 1 mm well combs

Weight 0.8g of powder and add it into a glass flask

Add to the glass flask 100mL of

Boil the suspension in a microwave for same minutes, until the solution will be completely clear

Add 10µL of FluoroVue 10000x nucleic acid gel stain and stir the solution until it will be completely homogeneous (agarose solution will become light yellow)

Add slowly the agarose solution to the gel tray and carefully remove possible bubbles

Let the agarose solution solidify for 0h 30m 0s at room temperature

In the meantime, prepare the unknown sample adding a standard volume of sample: 2.5µL (Comment: this volume corresponds to about 700ng of plasmidic DNA or RNA), 2µL of tartrazine tracking dye and 7.5µL of nuclease free water. Mix the sample by pipetting three or four times

Spin the sample to disrupt possible bubbles and keep the sample on ice until the loading

When the gel is solidified, setup the electrophoresis apparatus: remove the tray and set the gel into the electrophoresis chamber, add TAE 1x solution to the electrophoresis chamber, carefully remove 1 mm-well combs

Load 10µL of unknow sample to the gel

Run the agarose gel with TAE 1x buffer, at 80V for 0h 40m 0s

Check the results at UV-transilluminator

In a graduated cylinder prepare 1L of TAE 1x solution diluting TAE 50x stock solution with deionized water: 20mL of TAE 50x and 980mL of deionized water are required

Prepare the tray to add agarose solution, adding two 1 mm well combs

Weight 1.8g of agarose powder and add it into a glass flask

Add to the glass flask 100mL of TAE 1x

Boil the suspension in a microwave for same minutes, until the solution will be completely clear

Add 10µL of FluoroVue 10000x nucleic acid gel stain and stir the solution until it will be completely homogeneous (agarose solution will become light yellow)

Add slowly the agarose solution to the gel tray and carefully remove possible bubbles

Let the agarose solution solidify for 0h 30m 0s at room temperature; in the meantime, prepare the sample adding 2µL of Orange loading dye to 10µL of PCR product

Spin the sample to disrupt possible bubbles and keep the sample on ice until the loading

When the gel is solidified, setup the electrophoresis apparatus: remove the tray and set the gel into the electrophoresis chamber, add TAE 1x solution to the electrophoresis chamber, carefully remove 1 mm -well combs

Load 10µL of sample to the gel or 55µL of Gene Ruler 50 bp

Check the gel at UV-transilluminator

Thaw the unknown samples and spin to put the sample at the bottom of the 0.2mL tube. Keep the sample on ice for all the experience

Dilute the sample with 10µL of nuclease free water

Add 4µL 5x Prime Script Buffer and the same volume of Random 6 mers 100micromolar (µM)

Add 1µL of Oligo dT primer 50micromolar (µM)

Add 1µL of Prime Script RT Enzyme Mix 1. The final volume of 20µL will be reached. (Comment: enzyme must be stored in a refrigerated block, to avoid repeated freeze and thaw cycles)

Pipette up and down three or four times with a p20 pipette set at 20µL

Spin down the sample with a microcentrifuge

Incubate the sample within a thermal cycler set at 37°C for 0h 15m 0s for complementary DNA synthesis, then incubate at 85°C for 0h 5m 0s to denature the reverse transcriptase enzyme. After the thermic program the sample must keep on ice. (Note this is a stop point, if required sample can be store at -20°C for several days).

(Comment: during the thermic program of the reverse transcription, the preparation of PCR sample will be started).

Add at the bottom of 0.2 ml tube 16µL of nuclease free water

Add 1.5µL of each primers concentrated 50 ng/µl

Carefully add 1µL of DNA generated at point 1 and keep on ice

Add 1µL of Wonder Taq Polymerase. The final volume of 30µL will be reached. (Note for organizers and students: enzyme must be stored in a refrigerated block, to avoid repeated freeze and thaw cycles)

Pipette up and down three or four times with a p200 pipette set at 30µL 3

Spin down the sample with a microcentrifuge

Incubate the sample within a thermal cycler setting the following amplification program:

-95°C 0h 3m 0s initial denaturation and polymerase activation)

-95°C 0h 0m 15s denaturation phase)

-60°C``0h 0m 30s annealing)

-72°C 0h 0m 30s elongation)

-Steps 2, 3 and 4 were repeated for 25 cycles

At the end a final elongation phase of 72°C for 0h 5m 0s min was added. After the thermic program the sample must keep on ice. (Comment: this is a stop point, if required sample can be store at -20°C for several days)

10 µl of obtained PCR products, will be loaded on the 1.8Mass / % volume agarose gel

Thaw the sample and pipette up and down two or three times to mix the sample

Add 1µL of sample diluent (in our case nuclease free water) onto the optical sensor and click ‘read blank’

Clear the optical sensor with deionized water

Withdraw 1µL of the sample and add directly onto the optical sensor

Click ‘read sample’ and note the two absorbance values: A₂₆₀ and A₂₈₀

After 0.8Mass / % volume agarose gel visualization calculate sample concentration considering that:

plasmidic DNA: 1OD of absorbance at 260 nm correspond to 50 µg/ml; RNA: 1OD of absorbance at 260 nm correspond to 40 µg/ml

Using the value of absorbance at 280 nm calculate A₂₆₀/A₂₈₀ratio to determine purity of the sample