Team:USP-Brazil/Protocols
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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.
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