Team:Sheffield/LabProtocols
From 2014.igem.org
(Difference between revisions)
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<li>Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.</li> | <li>Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.</li> | ||
<li>Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.</li> | <li>Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="pageSection2"> | ||
+ | <div class="pageData"> | ||
+ | <h2>Protocol 4: Generate Chemically Competent E. Coli</h2> | ||
+ | <h3>Materials and Equipment</h3> | ||
+ | LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer. | ||
+ | <h3>Time</h3> | ||
+ | Prep: 40 minutes<br/> | ||
+ | Run: 5 hours | ||
+ | <h3>Procedure</h3> | ||
+ | <ol> | ||
+ | <li>Grow Cells | ||
+ | <ol> | ||
+ | <li>Take 1ml starter culture and add to 100ml LB broth.</li> | ||
+ | <li>Incubate at 37°C, 150rpm.</li> | ||
+ | <li>Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.</li> | ||
+ | <li>0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.</li> | ||
+ | <li>Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.</li> | ||
+ | </ol> | ||
+ | </li> | ||
</ol> | </ol> | ||
</div> | </div> |
Revision as of 07:35, 14 September 2014
Lab Protocols
Protocol 1: Produce LB Broth
Materials and Equipment
LB broth mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.Time
Prep: 5 minutesRun: 2 hours
Procedure
- Weigh out 2g of LB broth mix in a weighing boat and add into a 250ml conical flask.
- Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
- Swirl.
- Stopper the flask with cotton wool and foil.
- Label on autoclave tape. Format 'iGEM, Room Number'.
- Move the flask(s) to the autoclave to be sterilised.
Protocol 2: Produce LB Agar
Materials and Equipment
LB Agar mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.Time
Prep: 5 minutesRun: 2 hours
Procedure
- Weigh out 3.5g of LB agar mix in a weighing boat and add into a conical flask.
- Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
- Swirl.
- Stopper the flask with cotton wool and foil.
- Label on autoclave tape. Format 'iGEM, Room Number'.
- Move the flask(s) to the autoclave to be sterilised.
Protocol 3: Make Overnight Starter Cultures
Materials and Equipment
Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.Time
Prep: 10 minutesRun: 16 hours
Procedure
- Use a stripette to take 5ml of LB broth from a 250ml conical flask.
- Dispense into a falcon tube.
- Sterilise a metal loop in a flame.
- Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.
- Agitate.
- Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.
- Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.
Protocol 4: Generate Chemically Competent E. Coli
Materials and Equipment
LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.Time
Prep: 40 minutesRun: 5 hours
Procedure
- Grow Cells
- Take 1ml starter culture and add to 100ml LB broth.
- Incubate at 37°C, 150rpm.
- Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.
- 0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.
- Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.