Griffitts:Stock solutions

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Antibiotics

Ampicillin (40 mL)

• 1 g Ampicillin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Chloramphenicol (40 mL)

• 0.3 g Chloramphenicol
• 40 mL 50% Ethanol
  • Do not filter sterilize
  • Store in refrigerator

Gentamycin (40 mL)

• 0.3 g Gentamycin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Kanamycin (40 mL)

• 0.1 g Kanamycin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Neomycin (40 mL)

• 0.8 g Neomycin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Spectinomycin (40 mL)

• 0.8 g Spectinomycin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Streptomycin (40 mL)

• 2.0 g Streptomycin
• 40 mL ddH₂O
• Filter sterilize
  • Store in refrigerator

Tetracycline (40 mL)

• 0.1 g Tetracycline
• 40 mL 50% Ethanol
  • Do not filter sterilize
  • Store in refrigerator

Antibiotic Specifications

Antibiotic	Abbr.	Stripe	Stock	E. coli	S. meliloti
Ampicillin	Ap	BLACK	25 mg/mL	100 μg/mL (250X)	N/A
Chloramphenicol	Cm	BROWN	7.5 mg/mL	30 μg/mL (250X)	N/A
Gentamycin	Gm	PURPLE	7.5 mg/mL	3 μg/mL (2500X)	15 μg/mL (500X)
Hygromycin	Hy	(none)	5 mg/mL	20 μg/mL (250X)	20 μg/mL (250X)
Kanamycin	Km	YELLOW	7.5 mg/mL	30 μg/mL (250X)	N/A
Neomycin	Nm	GREEN	20 mg/mL	N/A	80 μg/mL (250X)
Spectinomycin	Sp	ORANGE	20 mg/mL	80 μg/mL (250X)	80 μg/mL (250X)
Streptomycin	Sm	BLUE	50 mg/mL	N/A	200 μg/mL (250X)
Tetracycline	Tc	RED	2.5 mg/mL	10 μg/mL (250X)	10 μg/mL (250X)

Note: These parameters are intended to be compatible with pre-made 250-mL amounts of LB agar, such that usually 1 mL of stock solution is diluted into the LB agar. For liquid LB (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exception: Gentamycin for E. coli.

DNA

10 mM dNTPs

• Thaw and mix (stir) 100 mM stock solutions of G, A, T, C (NEB)
• Combine in a 1.5-mL tube:
  • 60 μl H₂O
  • 10 μl G
  • 10 μl A
  • 10 μl T
  • 10 μl C

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

10X Taq buffer

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

10X Pfx buffer

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

Media Additives

1 M Calcium Chloride (75 mL)

• 75 mL ddH₂O
• 11.027 g CaCl₂·2H₂O

Autoclave

2% X-gal

• 10 mL N,N-dimethylformamide (DMF)(in fume hood)
• 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer)

Store at -20°C

0.2 M IPTG

• 5 mL ddH₂O
• 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)

Sterile filter, aliquot, and store at -20°C

20% SDS (75 mL)

• 75 mL ddH₂O
• 15 g Sodium dodecyl sulfate (SDS)

Do not sterilize

30% Acrylamide (75 mL)

• 75 mL ddH₂O
• 22.5 g acrylamide

10% Ammonium persulfate (75 mL)

• 75 mL ddH₂O
• 7.5 g ammonium persulfate (APS)

5 M NaCl (75 mL)

• 75 mL ddH₂O
• 21.915 g NaCl

Autoclave

80% Glycerol (75 mL)

• 75 mL ddH₂O
• 60 g glycerol

Note: use heating to mix
Autoclave

Orcinol spray

• 5 mL ddH₂O
• 25 mL methanol (in fume hood)
• 10 mL sulfuric acid (H₂SO₄ (in fume hood—be careful!)
• 200 mg orcinol