Lab Media Recipies

Davis Media

Potassium Phosphate Dibasic 7.0 g
Potassium Phosphate Monobasic 2.0 g
Sodium Citrate 0.5 g
Magnesium Sulfate Heptahydrate 0.1 g
Ammonium Sulfate 1.0 g
Thiamine pinch

Procedure:

• mix all chemicals in proper order
• Add 500 ml of water to mix and then
• add 500 ml of water to dilute.
• Autoclave in (1) 2L erlenmeyer flask or (2) 1 L flasks, or etc.
• Add 100 ml of water to glucose

LB Medium (Luria-Bertani Medium)

• Bacto tryptone 10 g
• Bacto yeast extract 5 g
• NaCl 10 g

Dissolve. Adjust pH to 7.0 with 5 n NaOH (several drops).

Yt Medium

• Bacto tryptone 8 g
• Bacto yeast extract 5 g
• NaCl 5 g

Dissolve. Adjust pH to 7.0 with 5 n NaOH (several drops).

2x Yt Medium (2YT)

• Bacto tryptone 16 g
• Bacto yeast extract 10 g
• NaCl 5 g

Dissolve. Adjust pH to 7.0 with 5 n NaOH (several drops).

Tryptone Broth (H Medium for Plate Lysates)

• Bacto tryptone 10 g
• NaCl 8 g

Use 12 g of agar per liter for plates.

R Medium for Phage Lysates

• Bacto tryptone 10g
• Bacto yeast extract 1 g
• NaCl 8 g

After autoclaving, add 2 ml of 1 m CaCl2 + 5 ml of 20% glucose. Use 12 g of agar per liter for plates.

Terrific Broth

• To 900 ml of H20, add
• Bacto tryptone 12g
• Bacto yeast extract 24 g
• glycerol 4 ml

Shake until the solutes have dissolved, and sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle. Allow the solution to cool to 60oC or less, and then add 100 ml of a sterile solution of 0.17 m KH2PO4. (This solution is made by dissolving 2.31 g of KH2PO4 and 12.54 g of K2HPO4 in 90 ml of H2O. After the salts have dissolved, adjust the volumes of the solution to 100 ml with H2O and sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle.)

SOB Medium

• To 950 ml of H2O, add
• Bacto tryptone 20 g
• Bacto yeast extract 5 g
• NaCl 0.5 g

Shake until the solutes have dissolved. Add 10 ml of a 250 mm solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 ml of H2O.) Adjust the pH to 7.0 with 5 n NaOH (~0.2 ml). Adjust the volume of the solution to 1 liter with H2O. Sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle. Just before use, add 5 ml of a sterile solution of 2 m MgCl2. (This solution is made by dissolving 19 g of MgCl2 in 90 ml of H2O, Adjust the volume of the solution to 100 ml with H2O and sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle.)

SOC Medium

SOC medium is identical to SOB medium except that it contains 20 mm glucose. After the SOB meduim has been autoclaved, allow it to cool to 60oC or less and then add 20 ml of a sterile 1 m solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 ml of H2O. After the sugar has dissolved, adjust the volume of the solution to 100 ml with H2O and sterilize by filtration through a 0.22-micron filter.)

Electrode Buffer (for running gels, horizontal and vertical)

• 9.08 g Tris Base
• 43.28 g Glycine
• 1.5 g SDS

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.
• Adjust ph to 8.30 using 10 M NaOH.

Stacking Gel Buffer (for vertical gels only)

• 9.08 g Tris Base
• 43.28 g Glycine
• 1.5 g SDS

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.
• Adjust ph to 6.79.

Staining Solution (for staining proteins, sera, enzymes, etc.)

• 250 ml MeOH
• 50 ml Glacial Acetic Acid
• 1 g Coomassie Blue
• 200 ml water

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.

Destaining Solution (for coomassie destaining)

• 100 ml MeOH
• 50 ml Glacial Acetic Acid
• 350 ml water

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.

Isopropanol (for preserving gels)

• 75 ml Isopropanol
• 425 ml water

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.

1% SDS

(I spray combs with this as a non-stick, also used when making vertical gel polyacrylamide gels, it levels the running gel before casting the stacking layer

• 1 g Sds
• 100 ml water

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.

The sera procedure is simple. Collect fresh animal blood, pig, sheep, cow, moose, caribou, etc. and centrifuge separating the sera from the whole cells. If the blood is not fresh the cells could undergo autolysis. Collect the sera in bullets (capped microfuge tubes - 1.5 mL) and freeze. It is handy to freeze the bullets in bullet racks, if not any plastic container with a lid works. Take one serum from each animal type. Make a dilution sera:running buffer of: 1:5, 1:10, 1:15, 1:20..... Record the dilution ratio for each animal type that gives a set of nice clean bands, no really concentrated bands, but still allows the fine lines to be seen. To do this the marker band needs to almost run off of the gels. You should then try to run a set of standards of each serum for your own info on a single gel, documenting the way the sera from each animal runs on your gels. This can be xeroxed after staining/destaining if placed between two acetate sheets and you can enlarge the run. You may need to play with the contrast a little. There are subtle differences between the sera, but the students should be able to tell the difference (although there are some sera that are more distinct than others). enough to identify an unknown serum when given the controls. You can make it more interesting if it is part of a detective scenario. You have four or five suspects. One is a known pig farmer, another a dairy man, another a chicken farmer, another a moose hunter, etc. You have samples of fresh blood extracted from their clothing and from the crime scene. Identify the murderer.

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