The David H. Koch Institute for Integrative Cancer Research at MITThe David H. Koch Institute for Integrative Cancer Research at MIT

Massachusetts Institute of Technology

National Cancer Institute Cancer Center

Science + Engineering... Conquering Cancer Together

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