Using the Autoclave
Interns can’t. Ask someone with a hard badge. Typical runs take about an hour, but could be two hours if the boiler isn’t warmed up.
Media
Most of the time you'll autoclave the media after putting it together, although certain chemicals (vitamins, antibiotics) need to be added after to prevent degradation.
LB: Use for E. coli
For 500mL, add:
5g Tryptone (or Peptone)
2.5g Yeast Extract
5g NaCl
7.5g Agar (if making plates)
Into 500ml of deionized water
AM (Acetobacter Media): Use for G. hansenii For 500mL, add:
10g Glucose
2.5g Tryptone (or Peptone)
2.5g Yeast Extract
1.35g Na2HPO4 0.75g Citric Acid
7.5g Agar (if making plates)
Into 500ml deionized water
Antibiotic Stocks
We usually make our liquid stocks of antibiotics at 1000X, so that you add 1μl/mL to whatever media you are using. While the desired working concentration might change based on the plasmid, here are the stock concentrations we usually use for common antibiotics:
Chloramphenicol: 34mg/mL (100% EtOH)
Ampicillin: 100mg/mL (50% EtOH)
Kanamycin: 20mg/mL (H20 only)
Neomycin: 50mg/mL (H20 only)
Tetracycline: 15mg/mL (50% EtOH)
You'll want to make these by filter sterilizing; you cannot autoclave antibiotics. We typically store them in 1mL aliquots in a -20C or -30C freezer. Those stocks made with ethanol will not freeze. Those in water only will require thaw time.
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