Team:Northern BC/Recipes
Northern BC iGEM 2019 Recipes
LB Media (Luria Broth)
LB media is used for growing bacterial cultures.
For liquid media: weigh the amount of LB powder indicated by the manufacturer (usually 20 - 25 g per litre) into a flask of appropriate size. Add the appropriate amount of water directly to the flask and swirl gently to dissolve the powder.
For large volumes, up to 1 L per flask, cover the flask with a double layer of aluminum foil and autoclave. Aluminum foil can be saved and reused. Remember to mark the flask with autoclave tape to ensure the media reached the appropriate temperature.
For smaller volumes, for example, 100 mL aliquots for bench use, pour 100 mL of media into 125 mL Wheaton bottles and autoclave.
For plates: weigh the amount of LB powder and agar indicated by the manufacturers (usually 20 - 25 g per litre) into a flask of appropriate size. Add the appropriate amount of water directly to the flask and swirl gently to dissolve the powder. Add a magnetic stir bar and autoclave (double tinfoil the top and mark with autoclave tape. Autoclave using the liquid setting and follow the time indicated on autoclave table [usually 15 min for up to 1 L]). Agar will not go into solution well until it has been heated.
Allow the media to cool on a stir plate with gentle stirring until you can safely handle the flask without burning yourself (keep tinfoil on). Do not let this go for too long as the agar will begin to solidify before the plates can be poured. General rule of thumb: the media is cool enough to proceed (~55 °C) when you can hold the palm of your hand against the side of the flask (where the media is; not at the neck) for 3 - 5 seconds without having to pull away (it should still feel uncomfortable, but not painful). Pour using aseptic technique. Flame the bottle neck each time you pour a plate and only open a plate for a few seconds. Stack plates in small piles on the side. You might want to work one hand with the media, one moving plates. Always keep the bottle neck near the flame. Put in enough media to cover the bottom of the plate (about half the plate – this is roughly 25 mL).
Leave plates alone for several hours to set. If you pour your plates late in the day they can sit overnight. LB plates should be bagged (label bag with type of plate, antibiotic, date, and your initials) and stored upside-down at 4 °C. 1 L makes two sleeves of plates.
Selective Media: Antibiotics are not stable in high heat and therefore will be destroyed if autoclaved. Add 1 mL of 1000X antibiotic per litre of media when the media has cooled enough to pour the plates as directed above. When adding antibiotic, remove tinfoil only briefly to add the antibiotic and then cover again until you are about to pour. Allow the media to stir for an additional minute to mix well and then proceed with plate pouring as above. Test plates by streaking a resistant strain, a non-resistant strain and nothing in 3 sections of a plate. Incubate for 16 h at 37 degrees and then check.
1000X (50 mg/mL) Carbenicillin
Carbenicillin is a stable ampicillin analog that is a powder stored at 4 °C. 1000X stock solutions can be prepared in 1.5 mL microcentrifuge tubes and stored at -20 °C. Label the tubes “1000X Carb”.
Prepare 10 mL of antibiotic stock solution (500 mg of carbenicillin in 10 mL ddH2O) in a 15 mL sterile conical tube and filter through a 0.45 um syringe filter. Store at -20 °C as 1 mL aliquots in sterile 1.5 mL microcentrifuge tubes.
Typical volumes used: 1 mL 1000X Carb in a 1 L culture (for preparing LB + Carb plates).
3 uL 1000X Carb in a 3 mL culture (for preparing overnight liquid
cultures).
Carbenicillin Disodium Salt (Ultrapure), VWR Cat# 97063-144
1000X (25 mg/mL) Chloramphenicol
Prepare 10 mL of antibiotic stock solution (250 mg of chloramphenicol in 10 mL ethanol) in a 15 mL sterile conical tube and filter through a 0.45 um syringe filter. Store at -20 °C as 1 mL aliquots in sterile 1.5 mL microcentrifuge tubes.
Typical volumes used: 1 mL 1000X Carb in a 1 L culture (for preparing LB + Carb plates).
3 uL 1000X Carb in a 3 mL culture (for preparing overnight liquid
cultures).
Yeast Media (YPD)
YPD is used to grow yeast without selection.
For 1 L of liquid YPD, combine in a flask of appropriate volume:
20 g Peptone (Difco Brand)
10 g Yeast Extract (Difco Brand)
20 g Dextrose (glucose)
Add 1 L of dH2O and autoclave.
For smaller volumes, for example, 100 mL aliquots for bench use, pour the media into 125 mL Wheaton bottles and autoclave.
For 1L of YPD Plates, mix the components listed above in a flask of appropriate volume. Add the appropriate amount of agar (usually 20 - 25 g per litre). Add a magnetic stir bar, cover the flask with a double layer of aluminum foil marked with autoclave tape and autoclave. Aluminum foil can be saved and reused.
Allow the media to cool on a stir plate with gentle stirring until you can safely handle the flask without burning yourself. Do not let this go for too long as the agar will begin to solidify before the plates can be poured. General rule of thumb: the media is cool enough to proceed (~55 °C) when you can hold the palm of your hand against the side of the flask (where the media is; not at the neck) for 3 - 5 seconds without having to pull away (it should still feel uncomfortable, but not painful).
Plates should be poured with a flame. Flame the neck of the flask before pouring each plate. Plates should be stacked on the benchtop and left overnight to set. YPD plates should be marked with the appropriate color code (see color code index in plate storage cabinet), bagged, and stored at room temperature in a closed cabinet.
1L makes two sleeves of plates.
1% Agarose Gel in 0.5X TBE with 0.5 µg/mL Ethidium Bromide
Weigh 1 g of Agarose into a 250 mL Erlenmeyer flask. Add 100 mL 0.5X TBE. Heat in the microwave until all agarose is dissolved. Allowed to cool for several minutes and then add 5 µL of 10 mg/mL ethidium bromide. Pour into casting stand, add the appropriate gel comb, and allow to solidify.
Agarose, Genetic Analysis Grade – Broad Separation Range for DNA/RNA –Fisher
Scientific, Cat# BP1356-100
Ethidium bromide, Bio-Rad Cat# 161-0-433
10X TBE Gel Running Buffer
For 1 L:Dissolve 107.8 g Tris Base (or Trizma Base from Sigma) (MW: 121.136 g/mol), 55 g Boric acid (MW: 61.83 g/mol) and 5.81 g EDTA (MW: 292.25 g/mol) in ~ 750 mL dH2O (or 7.4 g EDTA disodium salt (MW: 372 g/mol)). Adjust to 1 L in a 1 L graduated cylinder. Autoclave (seems to stay in solution better although this is not necessary for running buffer).
Tris Base
Boric Acid, Crystalline/Electrophoresis, Fisher BioReagents Cat# BP168-1
EDTA
50% Glycerol
To prepare 300 mL: Mix 150 mL of Glycerol (Glycerine) with 150 mL dH2O. Stir until completely mixed. Aliquot 100 mL per 125 mL Wheaton bottle and autoclave.
Glycerol (Molecular Biology Grade), Fisher BioReagents Cat# BP229-1
1 M Lithium Acetate
For 300 mL: Dissolve 30.61 g of Lithium acetate dihydrate (MW: 102.02 g/mol) in ~ 225 mL. Adjust to 300 mL in a 500 mL graduated cylinder. Aliquot 100 mL per 125 mL Wheaton bottle and autoclave.
Lithium acetate dihydrate, Reagent Grade, Fisher Scientific Cat# AA1341730
10X Tris-Cl Buffers
For 300 mL:Dissolve 36.34 g of TRIS (MW: 121.136 g/mol) in ~ 225 mL (called Trizma Base if ordered from Sigma-Aldrich).
pH to 6.8, 7.5, or 8.0 with HCl, starting with concentrated HCl (12.1 M) and finish with 1 M HCl as you get closer to the final pH.
Tris-HCl pH 8.0 is challenging to attain because it is on the edge of the buffering capacity. Take your time or you will overshoot and will need to start over.
Adjust to 300 mL in a 500 mL graduated cylinder.
Aliquot 100 mL per 125 mL Wheaton bottle and autoclave.
10X TE
Mix 10 mL 1 M Tris-Cl (desired pH), 10 mL 10 mM EDTA, and 80 mL ddH2O In a Wheaton bottle and autoclave.
5 M Sodium Chloride (NaCl)
To prepare 300 mL: Dissolve 87.66 g of anhydrous Sodium chloride (MW: 95.21 g/mol) in ~ 225 mL. Adjust to 300 mL in a 500 mL graduated cylinder. Aliquot 100 mL per 125 mL Wheaton bottle and autoclave.
