Common Lab Solutions

General notes about making solutions:

1. In general gloves, coat, and eye protection should be worn when making up solutions.  To avoid noxious fumes or spills, dispense concentrated acids and bases in the fume hood.  Only small volume container of acid and base solutions should be used at the pH meter.

1. Use clean baked (foil covered) beakers for mixing solutions (0.1 - 1L).  Smaller volumes solutions can be mixed in 15 or 50 mL sterile disposable conical tubes.  Large volume solutions (7 -8 L) are less precise and can be made directly in their carboy containers.

2. Use high quality filtered water for all laboratory solutions. 

3. Measure powders into a disposable weigh boat.  Use clean sterile spoons or scoopulas sparingly.  Discard excess material do not return it to the stock containers.

4. If solutions need to be pH'd then only add 80% of the volume of H2O needed prior to pHing to be sure that the final volume is not exceeded.

5. After all of the chemicals have been added transfer the solution to a 0.5 or 1.0 L graduated cylinder and q.s. with H2O. 

6. Divide (0.5 or 1L) solutions into sterile 500 mL media bottles prior to autoclaving (if directed).   Small volume solutions are sterilized by filtration with a sterile syringe and syringe tip 0.25 µm filters.  Larger volume solutions should be filtered on bottle top filters in a tissue culture hood if their ingredients are heat sensitive (e.g. tissue culture media).  

Ampicillin (50 mg/mL stock)

5 g. Ampicillin

100 mL H₂O

Filter sterilize in 1.5 mL aliquots

Store at -20°C

 

Bacteria plates (Makes 30 plates per 1 L)

1 L. LB media (See below)

15 g. Bacto-agar (for plates, use 7.5 gm for top agar)

Autoclave in a large (e.g. 2L flask)

Cool flask to 55°C in a water bath

Add antibiotics when media is < 55°C (not too hot to handle) 

Lay empty dishes on a benchtop.

Use a glove and quickly pour enough LB agar into each dish to cover the plate (or ~4 mm thick.)

Avoid tipping the flask repeatedly to mimimize foaming

Pass a Bunsen burner flame over open plates to pop air bubbles

Partially cover plates with their lids untill they cool off and harden

Leave plates at room temperature overnight to allow condensation to evaporate from lids

Stack plates and mark them to indicate the type of antibiotic.  Store them at 4ºC in the original plastic sleeve

Coomassie Blue Stain (1 L)

2.5 g. Coomassie brilliant blue  

500 mL Methanol

425 mL H₂O

75 mL glacial acetic acid

 

Destain (5 L)

500 mL Methanol

500 mL Glacial Acetic acid

4 L H₂O

 

   EDTA (0.5 M)

186.1 g. EDTA (disodium salt)

700 mL H₂O

pH to 8.0 with 10N NaOH (this helps to dissolve the EDTA)

q.s. 1 L. H₂O

Sterilize by autoclaving

 

   LB Medium (per 1 L)

10 g. Bacto-tryptone

5 g. Bacto-yeast extract

10 g. NaCl

Adjust pH to 7.5 with NaOH

q.s. H₂O

Sterilize by autoclaving

 

   5M NaCl

146 g NaCl

q.s. to 500 mL with H₂O

Sterilize by autoclaving

 

10x PBS, pH=7.4 (per 1 L)

1.44 g. KH2PO4 (Potassium Phosphate monobasic) (MW = 136 g/mole)

90 g. NaCl (MW = 58 g/mole)

7.95 g. Na2HPO4-7H2O (Sodium Phosphate dibasic) (MW = 268 g/mole)

q.s. to 1 L with H2O.

Sterilize by autoclaving.

SDS running buffer, 10x - make directly in 8 L carboy.

1.0 kg Glycine

140 g. Tris base

70 g. SDS

 

10% SDS

50 g. Sodium Lauryl sulfate (Sigma L-5750)

q.s. to 0.5 L. with H₂O

 

SSC, 20x (3M NaCl, 0.3M Citrate)

175 g NaCl

 88.2 g NaCitrate

 pH to 7.4 with NaOH or HCl

q.s. to 1 L. with H₂O

 

TBE, 5x (8 L) - make directly in 8 L carboy.

(Note: This is often used at 0.5x)

432 g Tris base

220 g Boric acid

37.2 g EDTA (disodium salt)

(or 160 mL 0.5 M EDTA)

q.s. 8 L. H₂O

TAE, 20x (6 L) - make directly in 8L carboy or 7L plastic jug.

581 g Tris base

137 g glacial acetic acid (17.4 M HOAc)  

240 mL 0.5 M EDTA

q.s. 6 L. H₂O

0.5% TNT (8 L) - make directly in 8 L carboy.

240 mL 5 M NaCl

100 mL 2M Tris pH7.5

400 mL 10 % v/v Tween 20 [0.5% v/v final]

7.26 L H₂O

 

Tris pH 7.4 (or pH 8.0)

60.5 g. Tris Base

400 mL H₂O

pH to 7.4 (or 8.0) with conc. HCl

q.s. to 500 mL with H₂O

 

   Tracking Dye, (20x)

15 mL Glycerol

18.3 mg bromphenol blue (BPB)

0.1 mL 1M Tris, pH8

Mix by inversion overnight

Aliquot into 1.5 mL centrifuge tubes

(Note: For a darker less viscous dye, Add 1 vol. T.E. and twice as much BPB)

 

Western Transfer Solution (A) - make directly in 8 L carboy.

24 g. Tris-base [25 mM final]

1.66 L Isopropanol [20% v/v final]

42.4 g. e-aminocaproic acid [40 mM final]

q.s. 8 L. H₂O

 

Western Transfer Solution (B) - make directly in 8 L carboy.

24 g. Tris-base [25 mM final]

1.66 L Isopropanol [20% v/v final]

q.s. 8 L. H₂O

 

Western Transfer Solution (C) - make directly in 8 L carboy.

242.4 g. Tris-base [250 mM final]

1.66 L. Isopropanol [20% v/v final]

q.s. 8 L. H₂O