Difference between revisions of "Team:Brasil-USP/Notebook/protocols"
Laisribovski (Talk | contribs) |
Laisribovski (Talk | contribs) |
||
| Line 100: | Line 100: | ||
<h1>Plasmid extraction </h1> | <h1>Plasmid extraction </h1> | ||
<br> | <br> | ||
| − | < | + | <ul> |
| − | < | + | <li>PureLink® Quick Plasmid Miniprep Kit-Life Technologies</li> |
| + | </ul> | ||
<br> | <br> | ||
<h2>Methodology</h2> | <h2>Methodology</h2> | ||
Revision as of 16:00, 16 September 2015
Protocols
Notebook
Protocols
Table of contents
- Circuit Assembly Protocols
- Calcium chloride transformation with heat shock in Escherichia coli DH5α
- Plasmid extraction
- Digestion of plasmidial DNA
- Ligation reaction (Cohesive ends)
- Agarose Gel Electrophoresis
- Directed mutagenesis PCR (for restriction site elimination) using a plasmid template for restriction site elimination
- PCR amplification (applied to lcp)
- PCR amplification for difficult amplicons (applied to roxA)
- Gibson assembly
- Characterization Protocols
...
Calcium chloride transformation with heat shock in Escherichia coli DH5α
Materials
- Sterile LB agar plate supplemented with the appropriate antibiotic (ampicillin 100 μg ml-1 or chloramphenicol 34 μg ml-1 - SIGMA-ALDRICH®);
- Sterile liquid LB media (SIGMA-ALDRICH®);
- Competent DH5α cells (Novagen) prepared through heat shock with calcium chloride;
- Plasmidial DNA.
Methodology
- Put the 0.5 mL microtube containing 50 μL competent cells aliquot on ice;
- Add 20-50 ng of plasmidial DNA or 10 μL of ligation reaction to the competent cells. Mix by pipetting carefully;
- Place the tube into a 42°C water bath for 2 min;
- Return the tube to the ice for 5 min;
- Add 200 μL of liquid LB;
- Incubate at 37°C, 250 rpm for 45 min;
- Plate the liquid LB containing the bacterial suspension on a LB agar plate with the appropriate antibiotic;
- Incubate overnight (14- 16h) at 37°C.
Plasmid extraction
- PureLink® Quick Plasmid Miniprep Kit-Life Technologies
Methodology
- Cell Growth
After isolating a single colony from a LB agar plate, grow it in 6 mL of liquid LB within the appropriate antibiotic. Incubate overnight (14-16h) at 37°C in a shaking incubator.
- Resuspension
Pellet the overnight culture in a 2 mL microtube and discard the supernatant. Repeat this step until the total liquid culture is finished. Resuspend the cell pellet in 240 μL of resuspension buffer by vortexing.
- Lysis
Add 250 μL of the Lysis buffer. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this period.
- Neutralization
Add 350 μL of the neutralization solution. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this time. Centrifuge at 16000 g for 10 minutes.
- Washing
ransfer the supernatant to a new 1.5 mL microtube with the resin. Be careful not to transfer the white pellet.
Add 650 μL of Wash buffer. Centrifuge at 16000 g for 1 minute. Discard the supernatant. Centrifuge again for 2-4 min to remove ethanol remains.
- Elution of plasmidial DNA
Put the resin in a new 1.5 mL microtube. Add 50 μL of nuclease free water at 65°C. Centrifuge at 16000 g for 3 minutes and discard the resin. Store DNA at -20°C.
- After isolating a single colony from a LB agar plate, grow it in 6 mL of liquid LB within the appropriate antibiotic. Incubate overnight (14-16h) at 37°C in a shaking incubator.
- Pellet the overnight culture in a 2 mL microtube and discard the supernatant. Repeat this step until the total liquid culture is finished. Resuspend the cell pellet in 240 μL of resuspension buffer by vortexing.
- Add 250 μL of the Lysis buffer. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this period.
- Add 350 μL of the neutralization solution. Mix by inversion 4-8 times and incubate at 37°C for 3-5 minutes. Do not exceed this time. Centrifuge at 16000 g for 10 minutes.
- ransfer the supernatant to a new 1.5 mL microtube with the resin. Be careful not to transfer the white pellet.
Add 650 μL of Wash buffer. Centrifuge at 16000 g for 1 minute. Discard the supernatant. Centrifuge again for 2-4 min to remove ethanol remains.
- Put the resin in a new 1.5 mL microtube. Add 50 μL of nuclease free water at 65°C. Centrifuge at 16000 g for 3 minutes and discard the resin. Store DNA at -20°C.