Methods

Following are recipes for all standard media prepared by the Media Preparation Facility.

3% Agar

  • 7.5g Bacto Agar/ 250 ml H2O

4% Agar

  • 10.0g Bacto Agar/ 250 ml H2O

40% Glucose

  • 400 g/l Glucose
  • autoclave

Hank's Balanced Salt Solution

Sigma # H-6136

  • 0.01 g/l Na2HPO4
  • 2.38 g/l Hepes
  • 0.35 g/l NaHCO3
  • adjust pH to 7.2
  • sterile filter (0.2 um)

Hepes-Buffered Saline

  • 7.07 g/l NaCl
  • 0.40 g/l KCl
  • 0.043 g/l Na2HPO4
  • 0.060 g/l KH2PO4
  • 1.00 g/l Glucose
  • 4.77 g/l Hepes
  • 0.0075 g/l Phenol red
  • adjust pH to 7.3
  • sterile filter (0.2 um)

LB Broth, Miller

  • 10.0 g/l Difco Bacto Tryptone
  • 5.0 g/l Difco Yeast Extrct
  • 10.0 g/l NaCl
  • 10.0 ml/l 1.0 M Tris pH 7.5
  • autoclave

2X LB Broth, Miller

  • double above g/l
  • autoclave

1.5% Agar/LB

  • 6.0 g Bacto Agar/ 400 ml LB

PBS

  • 8.0 g/l NaCl
  • 0.2 g/l KCl
  • 1.15 g/l Na2HPO4
  • 0.2 g/l KH2PO4
  • adjust pH to 7.2
  • autoclave

10X PBS

  • 80.0 g/l NaCl
  • 2.0 g/l KCl
  • 11.5 g/l Na2HO4
  • 2.0 g/l KH2PO4

Note: neither pH adjusted nor sterilized

2X S

  • 13.4 g/l Difco Bacto Yeast Nitrogen Base w/o amino acids
  • autoclave

2X S w/o ura, trp

  • 13.4 g/l Difco Bacto Yeast Nitrogen Base w/o amino acids
  • 4.0 g/l amino acid powder w/o ura, trp (prepared by Amon Lab)
  • autoclave

2X S w/o his, leu

  • 13.4 g/l Difco Bacto Yeast Nitrogen Base w/o amino acids
  • 4.0 g/l amino acid powder w/o his, leu (prepared by Amon Lab)

2X S w/o 6 aa

  • 13.4 g/l Difco Bacto Yeast Nitrogen Base w/o amino acids
  • 4.0 g/l amino acid powder w/o ade, his, leu, lys, trp,ura (prepared by Amon Lab)

Terrific Broth

Solution 1 (prepared by the Media Preparation Facility)

  • 12.0 g/l Difco Bacto Tryptone
  • 24.0 g/l Difco Bacto Yeast Extract or EMD Yeast Extract
  • 4.0 ml/l Glycerol
  • 900 ml H2O
  • autoclave

Solution 2 (prepared by the individual labs)

  • 23.1 g/l KH2PO4
  • 125.4 g/l K2HPO4
  • autoclave and cool
  • add 100 ml Solution 2 to 900 ml Solution 1

Versene

  • 8.0 g/l NaCl
  • 0.2 g/l KCl
  • 1.15 g/l Na2HPO4
  • 0.2 g/l KH2PO4
  • 0.2 g/l Na-EDTA
  • 0.015 g/l Phenol red
  • adjust pH to 7.2
  • autoclave

1.05X YEP

  • 10.5 g/l EMD Yeast Extract or Difco Bacto Yeast Extract
  • 21.1 g/l Difco Bacto Peptone
  • autoclave

2.22X YEP

  • 22.2 g/l EMD Yeast Extract or Difco Bacto Yeast Extract
  • 44.4 g/l Difco Bacto Peptone
  • autoclave

DME+/Hepes

SAFC Biosciences #56499C with: 4500 mg/l glucose, 110 mg/l Na pyruvate

  • 0.116 g/l L-arginine*HCl
  • 0.036 g/l L-asparagine
  • 0.216 g/l L-glutamine
  • 2.38 g/l Hepes
  • 2.0 g/l NaHCO3
  • Do NOT pH
  • sterile filter (0.2 um)

DME-LG

Gibco # 31600 with 1000 mg/l glucose and 110 mg/l Na pyruvate

  • 2.0 g/l NaHCO3
  • adjust pH to 7.15
  • sterile filter (0.2 um)

F-12 (Ham)

Gibco #21700

  • 2.0 g/l NaHCO3
  • adjust pH to 7.15
  • sterile filter (0.2 um)

MEM alpha

Gibco #12000

  • 2.2 g/l NaHCO3
  • adjust pH to 7.15
  • sterile filter (0.2 um)

MEM- (S-MEM, JOKLIKS)

Sigma # 56449C

  • 2.0 g/l NaHCO3
  • adjust pH to 7.2
  • sterile filter (0.2 um)

MEM w/Erle's Salts

Gibco # 41500 with: Earle's salts (non-essential amino acids, L-glutamine)

  • 1.5 g/l NaHCO3
  • adjust pH to 7.2
  • sterile filter (0.2 um)

RPMI

Gibco # 31800

  • 0.30 g/l Glutamine
  • 2.20 g/l NaHCO3
  • adjust pH to 7.15
  • sterile filter (0.2 um)

Pen/Strep

Mediatech #MT30-002-CI (VWR 45000-652)
10,000 IU/ml penicillin/10,000 ug/ml streptomycin

Trypsin/EDTA

  • 0.20 g/l EDTA
  • 8.00 g/l NaCl
  • 0.40 g/l KCl
  • 0.35 g/l NaHCO3
  • 1.00 g/l Glucose
  • 0.04 g/l Trypsin ( 428 units/ml) (Sigma # T-4665)
  • 0.0075 g/l Phenol red
  • adjust pH to 7.0
  • sterile filter (0.2 um)
  • Freeze immediately after filtering