Team:USP-Brazil/Protocols
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<p>3) Digest to linearize the plasmid</p> | <p>3) Digest to linearize the plasmid</p> | ||
+ | |||
+ | <p class="table"> | ||
+ | <b>Table 1:</b> Candidate promoters from in Pichia pastoris | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr class="tr_first"><td style="width:30%">Reagents</td><td style="width:30%">1 reaction</td></tr> | ||
+ | <tr><td>ECO RI*</td><td>1μL</td></tr> | ||
+ | <tr><td>Enzyme Buffer</td><td>2μL</td></tr> | ||
+ | <tr><td>DNA</td><td>2μL</td></tr> | ||
+ | <tr><td>H<sub>2</sub>O</td><td>15μL</td></tr> | ||
+ | <tr><td>Total</td><td>20μL</td></tr> | ||
+ | |||
+ | </table> | ||
+ | * place the enzyme last | ||
+ | |||
+ | <br></br> | ||
+ | <h4>Solutions P1, P2 and P3</h4> | ||
+ | |||
+ | <p>P1)</p> | ||
+ | <ul> | ||
+ | <li>23 ml glucose 20%</li> | ||
+ | <li>GTE 500 ml</li> | ||
+ | <li>10 ml EDTA 0.5 M pH 8,0</li> | ||
+ | <li>12.5 ml of Tris HCl 1M pH 7,0</li> | ||
+ | <li>Water to 500 ml</li> | ||
+ | <li>Autoclave before using</li> | ||
+ | </ul></p> | ||
+ | |||
+ | <p>P2)</p> | ||
+ | <ul> | ||
+ | <li>H2O 5580 ul</li> | ||
+ | <li>NaOH 120 ul</li> | ||
+ | <li>300 ul SDS 20%</li> | ||
+ | </ul></p> | ||
+ | |||
+ | <p>P3)</p> | ||
+ | <ul> | ||
+ | <li>KaOH 3M</li> | ||
+ | <li>60 ml KOAC 5M</li> | ||
+ | <li>11.5 ml Glacial Acetic Acid</li> | ||
+ | <li>Water to 100 ml</li> | ||
+ | <li>11.5 ml Glacial Acetic Acid</li> | ||
+ | <li>Autoclave before using</li> | ||
+ | </ul></p> | ||
+ | |||
+ | <h4>Protocol for Miniprep from Plate (used when there was a large amount of samples)</h4> | ||
+ | <ul> | ||
+ | <li>Take the plates out of the fridge</li> | ||
+ | <li>Add 1 µL of media (LB) with ampicillin (100 µg/mL) to each well | ||
+ | <li>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. | ||
+ | <li>Put on a shaker at 37°C at 300 RPM for 22 hours | ||
+ | <li>Prepare the 96-well plate with 80 µL with 50% glycerol | ||
+ | <li>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. | ||
+ | <li>Centrifuge at 4000 RPM for 10 minutes (put RNAse on ice to thaw) | ||
+ | <li>Discard the supernatant and turn plates upside down on paper towel | ||
+ | <li>Add 240 µL of solution 1; seal plates with [worse] cover and resuspend with vortex. | ||
+ | <li>Centrifuge at 4000 RPM for 10 minutes | ||
+ | <li>Prepare solution 2 | ||
+ | </ul></p> | ||
+ | |||
</div> | </div> | ||
</div> | </div> | ||
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Revision as of 22:19, 25 September 2013
Template:Https://2013.igem.org/Team:USP-Brazil/templateUP
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
- Prepare the 96-well plate with 80 µL with 50% glycerol
- 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.
- Centrifuge at 4000 RPM for 10 minutes (put RNAse on ice to thaw)
- Discard the supernatant and turn plates upside down on paper towel
- Add 240 µL of solution 1; seal plates with [worse] cover and resuspend with vortex.
- Centrifuge at 4000 RPM for 10 minutes
- Prepare solution 2