Team:USP-Brazil/Protocols

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Problem

Protocols

Cloning

Plasmid DNA isolation

Extraction of plasmid DNA from E. coli by Alkaline Lysis – Miniprep

1.1) Formation of pellets*

  • Transfer 1.5 ml of E. coli sample to eppendorf.
  • Centrifuge at 14000 RPM for one minute at 4°C.
  • Discard the supernatant.
  • Add the remaining E. coli, centrifuge again and discard the supernatant.
  • Resuspend in 200 μL of Solution 1 (P1) and vortex to dilute the entire pellet.
*: Keep all on ice

1.2) Extraction of DNA

  • Add 200 μL of Solution 2 (P2) freshly made.

Solution 2*:
For 2 mL: 1860 μL of milliQ water, 40 μL of NaOH 10N, and 100 μL of SDS 20%.
For 5 mL: 4650 μL of milliQ water, 100 μL of NaOH 10N, and 250 μL of SDS 20%.
*: The reagents MUST be added in the order described. Stir after each mix. Do not put ice or you might have to redo if a precipitate (crystal) forms.

  • Gently invert 6 times.
  • Leave at room temperature for 5 minutes.
  • Add 200 μL of Solution 3 (P3), flip and put on ice immediately.
  • Centrifuge at 14000 RPM for 20 minutes at 4°C.
  • Collect the supernatant (with pipette) without getting any of the precipitate pellet moved into the new eppendorf. Collect 200 μL at a time with the pipette tip to prevent getting any of the pellet.

1.3) Precipitation of DNA

  • Add 400 μL of isopropanol. Invert 5 times.
  • Connect the speed vaccum.
  • Centrifuge at 14000 RPM for 10 minutes at 4°C.
  • Discard the supernatant and invert eppendorf over paper towel.

1.4) Dry DNA

  • Add 500 μL of ethanol 70%. Invert 5 times.
  • Centrifuge at 14000 RPM for 5 minutes at 4°C.
  • Discard and speed vacuum for 15 minutes.

1.5) Resuspend the DNA and degrade the RNA

  • Resuspend in 30 μL of milliQ water with light finger touches.
  • Treat with 5 μL RNAse per sample. Resuspend with light finger touches.
  • Leave at 37°C for at least half an hour.
  • Place in 20°C freezer for overnight storage.

2) Quantification by Nanodrop

  • Connect software.
  • Select option “Nucleic Acid.”
  • Place 2 μL of milliQ water on each pedestal and clean with paper towel.
  • Place 2 μL of milliQ water to calibrate the instrument and press “OK.”
  • Carry out the “Blank” using 2 μL of either milliQ water or buffer, depending on what you used to dilute your DNA sample.
  • Place 2 μL of sample and click measure.
  • Observe the quantification of DNA in ng/μL.
  • Also observe the ratio of 260/280, which must be around or greater than 1.7-1.8.

3) Digest to linearize the plasmid