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
Table 1: Candidate promoters from in Pichia pastoris
Reagents | 1 reaction |
ECO RI* | 1μL |
Enzyme Buffer | 2μL |
DNA | 2μL |
H2O | 15μL |
Total | 20μL |
Solutions P1, P2 and P3
P1)
- 23 ml glucose 20%
- GTE 500 ml
- 10 ml EDTA 0.5 M pH 8,0
- 12.5 ml of Tris HCl 1M pH 7,0
- Water to 500 ml
- Autoclave before using
P2)
- H2O 5580 ul
- NaOH 120 ul
- 300 ul SDS 20%
P3)
- KaOH 3M
- 60 ml KOAC 5M
- 11.5 ml Glacial Acetic Acid
- Water to 100 ml
- 11.5 ml Glacial Acetic Acid
- Autoclave before using
Protocol for Miniprep from Plate (used when there was a large amount of samples)
- Take the plates out of the fridge
- Add 1 µL of media (LB) with ampicillin (100 µg/mL) to each well
- Choose colonies by poking with a sterile toothpick and inoculate (put toothpick in well). Cover the 96-well plate with adhesive cover (the worse version) and poke a hole through cover with a needle.
- Put on a shaker at 37°C at 300 RPM for 22 hours
- [in sterile hood] Prepare the 96-well plate with 80 µL with 50% glycerol
- [in sterile hood] Pipette 80 µL of bacteria and mix with a pipette with the glycerol. Cover the 96-well plate with (better) adhesive cover along with plastic cover. Place glycerol stock in -70°C freezer.
- [on ice] Centrifuge at 4000 RPM for 10 minutes (put RNAse on ice to thaw)
- [on ice] Discard the supernatant and turn plates upside down on paper towel
- [on ice] Add 240 µL of solution 1; seal plates with [worse] cover and resuspend with vortex.
- [on ice] Centrifuge at 4000 RPM for 10 minutes
- [on ice] Prepare solution 2
- [on ice] Discard the supernatant and turn plates upside down on paper towel
- [on ice] Add 80 µL of solution 1 – seal plate and resuspend cells with vortex
- [on ice] Take the U-bottom 96-well plate, and add 5 µL of RNAse (10 mg/mL) per well
- Transfer 60 µL of cells to the U-bottomed plate
- Add 60 µL of solution 2; seal with [better] cover and invert plate 10 times
- Leave on the bench for 10 minutes. Spin for a few seconds. (Turn on and preheat heater to 80°C)
- Add 60 µL of solution 3. Seal with [better] cover and invert 10 times
- Leave on the bench for 10 minutes. Spin for a few seconds.
- Take off cover and place plate in 80°C heater.
- [no adhesive cover] Put on ice for 10 minutes.
- [no adhesive cover] Centrifuge at 4000 RPM for 10 minutes
- [no adhesive cover] Attach V-bottom 96-well plate to plate with filter and secure using masking tape
- Transfer all the sample without the pellet and centrifuge at 4000 RPM for 15 minutes.
- Discard the filter plate and add 110 µL of cold 100% isopropanol
- Cover the plate with [better] cover and invert 10 to 20 times
- Spin, change the seal to another cover and centrifuge for 45 minutes (turn on speed vacuum)
- Discard the supernatant and add 200 µL of cold 70% ethanol
- Centrifuge for 5 minutes at 4000 RPM. Discard supernatant
- Dry the plate for 15 minutes using the speed vacuum
- Resuspend with 40 µL of water