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Pre-Labs

Week Supplies Needed Pre-lab (day 0) Pre-lab (day 1) Pre-lab (day 2) Notes

1

  • food coloring
    (5 colors)
  • 1.5-ml tubes
  • 15-ml culture tubes
  • 50-ml conical tubes
  • VALAP
  • microscope slides

Prepare:

  • overnight culture (enough for microscope slide for each group)

Prepare:

  • beaker for tip disposal
  • VALAP

Prepare (for each group):

  • four 1.5-ml tubes (1 ml each of colored solutions I, II, III, IV)
  • one 50-ml conical tube (25 ml of colored solution V)

afternoon labs only

2

  • E. coli plate
  • LB broth
  • 50-ml conical tubes
  • cuvettes

Autoclave:

  • culture tubes
  • Erlenmeyer flasks

Prepare (if not ready):

  • E. coli plate

Set:

  • incubator to 37°C

Set:

  • incubator to 37°C
  • spectrophotometer for use
3
  • agarose
  • EtBr (or SYBR Safe)
  • λ DNA
  • EcoRI, BamHI, HindIII
  • loading dye
  • 2x restriction buffer
  • TBE buffer

Set:

  • water bath to 37°C

Prepare (for each group):

  • 20 μ of 0.1 μg/μl λ DNA (on ice)
  • 25 μ of 2x restriction buffer (on ice)
  • 2 μ each of EcoRI, BamHI, HindIII (on ice)
  • 500 μl of distilled water
  • 500 μl of loading dye
  • 500 ml 1x TBE buffer
  • EtBr solution

Clean:

  • gel boxes
  • staining trays

afternoon only

4

  • E. coli slant or plate
  • 15-ml culture tubes
  • plates
  • pGREEN plasmid
  • LB agar
  • LB broth
  • CaCl2 solution
  • ampicillin

Autoclave:

  • CaCl2 solution
  • LB broth

Prepare:

  • several "starter plates" of E. coli

Prepare (for each group):

  • two LB agar plates
  • two LB+ampicillin plates

Set:

  • water bath to 42°C
  • incubator to 37°C

Prepare:

  • 95% ethanol in beaker covered with Parafilm

Prepare (for each group):

  • 1 ml of sterile 50 mM CaCl2 in a 1.5-ml tube (on ice)
  • 1 ml of sterile LB broth in a 1.5-ml tube
  • 12 μl of 0.005 μg/μl pGREEN in a 1.5-ml tube (on ice)

Set:

  • camera & blacklight out

5

  • E. coli slant or plate
  • E. coli/pAMP slant or plate
  • 15-ml culture tubes
  • LB broth
  • LB broth/ampicillin
  • Penicillin-G
  • 0.04% phenol red solution

Prepare:

  • 5 mL E. coli overnight culture in 15-mL tube
  • 5 mL E. coli/pAMP overnight culture in 15-mL tube

Prepare (for each group):

  • 5 mL E. coli overnight culture
    (in 15-mL tube labeled "control")
  • 5 mL E. coli/pAMP overnight culture
    (in 15-mL tube labeled "pAMP")
  • 6 mL of 4 mg/mL penicillin-G
  • 1 mL of 0.04% phenol red solution

6

7

  • E. coli slant or plate
  • pAMP (0.005 ug/uL)
  • pKAN (0.005 ug/uL)
  • pLIG (ligated pAMP and pKAN from DNA Science Lab 9)
  • LB
  • LB/amp plates
  • LB/kan plates
  • LB/amp+kan plates
  • CaCl2 (50 mM)
  • 1.5 mL and 15 mL tubes

Start overnight culture.
Prepare (for each group):

  • 3 LB/amp plates
  • 3 LB/kan plates
  • 3 LB/amp+kan plates

Autoclave (if not sterile):

  • 1.5 mL tubes
  • 15 mL tubes
  • CaCl2
  • LB

Start mid-log culture.
Set:

  • Water bath to 42C
  • Shaker-incubator to 37C
  • Incubator to 37C (make sure there is room for 9 plates/experiment)

Prepare (for each group):

  • Crushed ice
  • 15 uL pAMP (0.005 ug/uL)
  • 15 uL pKAN (0.005 ug/uL)
  • 4.5 mL CaCl2
  • 3 mL LB

Prepare:

  • Space in the refrigerator for 9 plates/experiment.

8

9

  • amp+kan resistant E. coli from week 7 or 8
  • sterile 1.5 mL and 50 mL tubes
  • sterile 1-10 uL and 100-1000 uL pipette tips
  • Miniprep reagents:
    • GTE
    • 10% SDS
    • 4 N NaOH
    • KOAc
    • Isopropanol
    • 95% or 100% ethanol
    • TE
  • Gel electrophoresis supplies:
    • 0.8% agarose gel
    • 1x TBE buffer
    • Loading dye
  • Enzymes:
    • BamHI (10 units/uL)
    • HindIII (10 units/uL)
    • RNase A (5 mg/mL)
    • 10x restriction buffer
  • Plasmids:
    • pAMP (0.1 ug/uL)
    • pKAN (0.1 ug/uL)
    • lambda DNA (0.1 ug/uL) or 1kb ladder

Sterilize:

  • 1.5 mL culture tubes
  • 1-10 uL pipette tips
  • 100-1000 uL pipette tips

Prepare for each group:

  • Overnight cultures of two amp+kan colonies

Aliquot for each group:

  • 250 uL GTE
  • 500 uL SDS/NaOH
    • 1 mL 10% SDS
    • 0.5 mL 4 N NaOH
    • 8.5 mL DI H2O
  • 400 uL KOAc
  • 1000 uL isopropanol
  • 500 uL ethanol
  • 40 uL TE
  • Set water bath to 37 C
  • Prepare for each group:
    • 50-100 mL 0.8% agarose gel
    • 500 mL 1x TBE
  • Aliquot for each group:
    • 5 uL pAMP/pKAN (1:1, 0.1 ug/uL)
    • 5 uL 1kb ladder (0.1 ug/uL)
      • 1 uL 1 kb ladder (0.5 ug/uL)
      • 4 uL TE buffer
    • 14 uL 5x Restriction Buffer/RNase A
      • 500 uL 10x restriction buffer
      • 100 uL RNase (5 mg/mL)
      • 400 uL DI H2O
    • 8 uL BanHI/HindIII (1:1, 10 units/uL)
    • 30 uL HI H2O
    • 8 uL loading dye
  • Optional lambda gel:
    • 6 uL lambda DNA (0.1 ug/uL)
    • 2 uL HindIII (10 units/uL)

10


Supply List

  • safety glasses
  • gloves - 2 boxes each (S, M, L)
  • plates
  • Erlenmeyer flasks - 12 (500 mL)