3.3 Salk phenotypes

3.3 Salk phenotypes margaret Wed, 11/16/2011 - 17:18

Change the lab manual so it says they have to follow any homozygous or heterozygous Salk plants throughout their entire life - till the end of the semester.

M 3/5/12

Per Pair:

  • Control seeds (aliquot ~25 into 12 microfuge tubes, dry)

  • Salk-line seeds (remainder from Lab 3.2, dry, ~25 per tube)

  • 2 mL 70% EtOH, 0.05% TritonX-100 (stock is 10%)

    • Make 50 mL, aliquot into microfuge tubes

    • 37 mL 95% EtOH

    • 0.25 mL 10% Triton-X
    • water to 50 mL

  • If seeds are to be plated in agar:

    • 0.1% agar, sterile
    • ~ 2 mL per pair
    • 0.1 g agar/100 mL, autoclave, aliquot.
    • Or aliquot into glass tubes, then autoclave

  • sterile water

  • If seeds are to be dried and plated from filter paper:

    • Filter paper (? 4 in a plastic Petri dish?)
    • Sterile forceps in tubes for handling filter paper
    • 10 mL 95% EtOH (aliquot 25 15mL tubes)

  • 2 complete medium plates per pair

  • 2 Low-Fe medium plates (1 µM Fe) per pair

  • micropore tape

Total plates (actually need 20 of each type)

  • 24 complete MS medium

  • 24 low Fe MS medium

salt-germination-10.xlsx

MS plates

MS plates kdorfman Mon, 12/19/2011 - 20:27

Murashige & Skoog Medium

30 mL per plate

Use the tall petri plates so the seedlings have more space

MS complete

MS complete kdorfman Mon, 12/19/2011 - 20:45
ingredient 750 800 1000 1200 1600 mL
number of plates 25 27 33 40 53
ddH2O* 450 480 600 720 960 mL
10X macronutrients 1 75 80 100 120 160 mL
10X micronutrients 2 75 80 100 120 160 mL
MES 0.375 0.4 0.5 0.6 0.8 g

*Add the other salt mixtures to the water to prevent precipitation

pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs ~720 µL/L)

Bring to volume with distilled water

ingredient 750 800 1000 1200 1600 mL
bacto or phyto agar 7.5 8 10 12 16 g

autoclave with stir bar in flask or bottle

Stir until cool enough to handle

Pour 30 mL per plate (use the deep ones)

(Or Sigma M5524 - macro and micro nutrients together.)

  1. Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26 ↩︎

  2. Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26 ↩︎

MS nutrients

MS nutrients kdorfman Mon, 02/13/2012 - 19:56

MS macronutrients 10X

Sigma M 0654

Component mg/L
Potassium phosphate monobasic 170
Magnesium sulfate 180.7
Calcium chloride anhydrous 332.2
Ammonium nitrate 1650
Potassium nitrate 1900

MS micronutrients 10X

Sigma M 0529

Component mg/L
Boric acid 6.2
Cobalt chloride . 6H2O 0.025
Cupric sulfate. 5H2O 0.025
Ferrous sulfate.7H2O 27.8
Manganese sulfate. H2O 16.9
Molybdic acid (sodium salt) o 2H2O 0.25
Na2-EDTA 37.3
Potassium iodide 0.83
Zinc sulfate.7H2O 8.6

MS low iron

MS low iron kdorfman Mon, 12/19/2011 - 21:12

**Murashige & Skoog 1µM iron medium

MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM

FeSO4 stock is 10 mM, which is 10,000x 1 µM

To make plates low-iron MS
start with mL ddH2O (~ 60% final volume)
add: mL 10X macronutrients
add: mL boric acid 1000X
add: mL cobalt chloride 10,000X
add: mL cupric sulfate 10,000X
add: mL ferrous sulfate 10 mM
add: mL KI 10,000X
add: mL manganese sulfate 1000X
add: mL moybdic acid 10,000X
add: mL zinc sulfate 1000X
add: g MES, pH to 5.7 with KOH
bring to mL final volume
add: g bacto- or phyto-agar
autoclave for minutes

ingredient 750 1000 1200 1500 mL
number of plates 25 33 40 50
ddH2O* 450 600 600 960 mL
10X macronutrients 75 100 120 150 mL
boric acid 1000x 0.75 1.0 1.2 1.5 mL
Cobalt chloride 10,000x 0.075 0.1 0.12 0.15 mL
cupric sulfate 10,000x 0.075 0.1 0.12 0.15 mL
ferrous sulfate 10,000x 0.075 0.1 0.12 0.15 mL
KI 10,000x 0.075 0.1 0.12 0.15 mL
manganese sulfate 1000x 0.75 1.0 1.2 1.5 mL
molybdic acid 10,000x x 0.075 0.1 0.12 0.15 mL
zinc sulfate 1000x 0.75 1.0 1.2 1.5 mL
MES 0.375 0.5 0.6 0.75 g

*Add the other salt mixtures to the water to prevent precipitation

pH to 5.7 with 1M KOH (initial pH = ~4.6) (needs ~570 drops/L)

Bring to volume with distilled water

ingredient 750 1000 1200 1500 mL
bacto or phyto agar 7.5 10 12 15 g

autoclave with stir bar in flask or bottle

Stir until cool enough to handle

Pour 30 mL per plate (use the deep ones)