Griffitts:Stock solutions

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==DNA==
==DNA==
===10 mM dNTPs===
===10 mM dNTPs===
-
* Thaw and mix (stir) 100 mM stock solutions of G, A, T, C (NEB)
+
* Thaw and mix (stir) 100 mM stock solutions of dGTP, dATP, dTTP, dCTP (NEB; in freezer)
* Combine in a 1.5-mL tube:
* Combine in a 1.5-mL tube:
** 60 μl H<sub>2</sub>O
** 60 μl H<sub>2</sub>O
-
** 10 μl [[Wikipedia:Guanine|G]]
+
** 10 μl [[Wikipedia:Guanine|dGTP]]
-
** 10 μl [[Wikipedia:Adenine|A]]
+
** 10 μl [[Wikipedia:Adenine|dATP]]
-
** 10 μl [[Wikipedia:Thymine|T]]
+
** 10 μl [[Wikipedia:Thymine|dTTP]]
-
** 10 μl [[Wikipedia:Cytosine|C]]
+
** 10 μl [[Wikipedia:Cytosine|dCTP]]
-
Note: Store in the -20°C freezer in the Box marked "Other Enzymes"
+
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
===10X ''Taq'' buffer===
===10X ''Taq'' buffer===
-
Note: Store in the -20°C freezer in the Box marked "Other Enzymes"
+
* Label tubes: 10X ''Taq'' buffer
 +
* Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
 +
* Aliquot 375 μL of "Thermo pol buffer" into tubes&mdash;do '''not''' dilute!
 +
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
===10X ''Pfx'' buffer===
===10X ''Pfx'' buffer===
-
Note: Store in the -20°C freezer in the Box marked "Other Enzymes"
+
Note: Store in the -20°C freezer in the box marked "Other Enzymes"
==Media Additives==
==Media Additives==

Revision as of 13:16, 2 April 2008

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Contents

Antibiotics

Ampicillin (40 mL)

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

Chloramphenicol (40 mL)

Erythromycin (20 mL)

  • 1.0 g Erythromycin
  • 20 mL 100% ethanol (in fume hood)
  • Do not filter sterilize—the ethanol may dissolve the filter
    • Store in the -20°C Freezer

Gentamycin (40 mL)

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

Hygromycin (40 mL)

Kanamycin (40 mL)

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

Nalidixic acid (30 mL)

  • 0.9 g Nalidixic Acid
  • 0.36 g NaOH
  • 30 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Neomycin (40 mL)

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

Rifampicin (20 mL)

  • 1.0 g Rifampicin
  • 20 mL N,N-dimethylformamide (DMF; in fume hood)
  • Do not filter sterilize—the DMF will dissolve the filter
    • Store in the -20°C Freezer

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)

Antibiotics Table

Antibiotic Specifications
Antibiotic Abbr. Stripe Stock E. coli S. meliloti Solvent Storage
Ampicillin Ap BLACK 25 mg/mL 100 μg/mL (250X) N/A ddH2O 4°C
Chloramphenicol Cm BROWN 7.5 mg/mL 30 μg/mL (250X) N/A EtOH 4°C
Erythromycin Em (none) 50 mg/mL N/A N/A EtOH –20°C
Gentamycin Gm PURPLE 7.5 mg/mL 3 μg/mL (2500X) 15 μg/mL (500X) ddH2O 4°C
Hygromycin Hy (none) 5 mg/mL 20 μg/mL (250X) 20 μg/mL (250X) ddH2O 4°C
Kanamycin Km YELLOW 7.5 mg/mL 30 μg/mL (250X) N/A ddH2O 4°C
Nalidixic acid Na (none) 5 mg/mL 30 μg/mL (1000X) N/A ddH2O 4°C
Neomycin Nm GREEN 20 mg/mL N/A 80 μg/mL (250X) ddH2O 4°C
Rifampicin Rm (none) 50 mg/mL N/A N/A DMF –20°C
Spectinomycin Sp ORANGE 20 mg/mL 80 μg/mL (250X) 80 μg/mL (250X) ddH2O 4°C
Streptomycin Sm BLUE 50 mg/mL N/A 200 μg/mL (250X) ddH2O 4°C
Tetracycline Tc RED 2.5 mg/mL 10 μg/mL (250X) 10 μg/mL (250X) EtOH 4°C
Triclosan (none) N/A N/A EtOH 4°C

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 dGTP, dATP, dTTP, dCTP (NEB; in freezer)
  • Combine in a 1.5-mL tube:

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

10X Taq buffer

  • Label tubes: 10X Taq buffer
  • Thaw and mix (stir) "Thermo pol buffer" (NEB; in freezer)
  • Aliquot 375 μL of "Thermo pol buffer" into tubes—do not dilute!

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

Copper sulfate

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
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