Media formula: Difference between revisions
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To make one liter of TAP: | To make one liter of TAP: | ||
*10 | *10 mL 2M Tris-acetate (pH 7.0) | ||
*10 | *10 mL Phosphate buffer (pH 7.0) | ||
*10 | *10 mL nutrient stock | ||
*10 | *10 mL trace metals (10x dilution) | ||
*Bring to 1 liter with distilled water | *Bring to 1 liter with distilled water | ||
===Variations=== | ===Variations=== | ||
| Line 13: | Line 13: | ||
===2M Tris-acetate stock=== | ===2M Tris-acetate stock=== | ||
*242 g Tris base | *242 g Tris base | ||
*Dissolve in 600 | *Dissolve in 600 mL water while titrating to pH 7.0 with glacial acetic acid. | ||
*Bring to 1 liter | *Bring to 1 liter | ||
===Phosphate buffer=== | ===Phosphate buffer=== | ||
| Line 19: | Line 19: | ||
*5.6 g KH<sub>2</sub>PO<sub>4</sub> | *5.6 g KH<sub>2</sub>PO<sub>4</sub> | ||
*Bring to 1 liter | *Bring to 1 liter | ||
=== | ===Nutrient stock=== | ||
*40 g NH<sub>4</sub>Cl | *40 g NH<sub>4</sub>Cl | ||
*10 g MgSO<sub>4</sub> | *10 g MgSO<sub>4</sub>·7H<sub>2</sub>O | ||
*5 g CaCl<sub>2</sub> | *5 g CaCl<sub>2</sub>·2H<sub>2</sub>O | ||
*Bring to 1 liter | *Bring to 1 liter | ||
=== | ===Trace metals=== | ||
Making this is a complex proceedure so (unless you think you will be using a lot of it) there are a couple of alternatives. | Making this is a complex proceedure so (unless you think you will be using a lot of it) there are a couple of alternatives. | ||
*Make a soil extract by shaking a few grams of soil in water | *Make a soil extract by shaking a few grams of soil in water | ||
| Line 30: | Line 30: | ||
*Use tap water rather than distilled. | *Use tap water rather than distilled. | ||
This makes a | This makes a liter of full-strength, enough for 1000 L of TAP. Dilute an aliquot tenfold and use the diluted stock in making TAP | ||
#Dissolve 50g of acid free EDTA in 250 ml of ddH<sub>2</sub>O. Heat to dissolve. | #Dissolve 50g of acid free EDTA in 250 ml of ddH<sub>2</sub>O. Heat to dissolve. | ||
#Dissolve the following, one by one, in order, heating to approximately 100° in 550 ml ddH<sub>2</sub>O. | #Dissolve the following, one by one, in order, heating to approximately 100° in 550 ml ddH<sub>2</sub>O. | ||
#* | #*11.40 g BO<sub>3</sub>H<sub>3</sub> | ||
#*ZnSO<sub>4</sub>·7H<sub>2</sub>O | #*22.00 g ZnSO<sub>4</sub>·7H<sub>2</sub>O | ||
#*MnCl<sub>2</sub>·4H<sub>2</sub>O | #*5.06 g MnCl<sub>2</sub>·4H<sub>2</sub>O | ||
#*FeSO<sub>4</sub>·7H<sub>2</sub>O | #*4.99 g FeSO<sub>4</sub>·7H<sub>2</sub>O | ||
#*CoCl<sub>2</sub>·6H<sub>2</sub>O 1. | #*1.61 g CoCl<sub>2</sub>·6H<sub>2</sub>O | ||
#*1.57 g CuSO<sub>4</sub>·5H<sub>2</sub>O | |||
#*1.1 g Mo<sub>7</sub>O<sub>24</sub>(NH<sub>4</sub>)<sub>6</sub>·4H<sub>2</sub>O (Ammonium Molybdate) | |||
#Mix the two solutions ( | #Mix the two solutions (1 & 2) together. The resulting solution should be blue-green. | ||
#Heat to 100°C. Cool slightly, but don't let the temperature drop below 80°-90°. | #Heat to 100°C. Cool slightly, but don't let the temperature drop below 80°-90°. | ||
#Adjust pH to 6.5 to 6.8 with 20% KOH (approximately 83 ml). Don't let the temperature drop below | #Adjust pH to 6.5 to 6.8 with 20% KOH (approximately 83 ml). Don't let the temperature drop below 70° until after the pH is adjusted. | ||
#Make up to 1L and let stand in a 2L Erlenmeyer flask, stoppered loosly. The color should change from green to purple over a period of days. | #Make up to 1L and let stand in a 2L Erlenmeyer flask, stoppered loosly. The color should change from green to purple over a period of days. | ||
#Remove the rust-coloured precipitate by filtering, with suction, through 3 layers of Whatman #1 filter paper in a Buchner funnel. Repeat until no more precipitate is seen on the filter paper | #Remove the rust-coloured precipitate by filtering, with suction, through 3 layers of Whatman #1 filter paper in a Buchner funnel. Repeat until no more precipitate is seen on the filter paper | ||
Latest revision as of 12:45, 5 August 2005
TAP (Tris-Acetate Phosphate)
To make one liter of TAP:
- 10 mL 2M Tris-acetate (pH 7.0)
- 10 mL Phosphate buffer (pH 7.0)
- 10 mL nutrient stock
- 10 mL trace metals (10x dilution)
- Bring to 1 liter with distilled water
Variations
- For plates add 15g agar per liter (1.5%)
- For TAP-sulfur substitute MgCl2 for the MgSO4 in the nutrient stock and omit trace metals
Materials
2M Tris-acetate stock
- 242 g Tris base
- Dissolve in 600 mL water while titrating to pH 7.0 with glacial acetic acid.
- Bring to 1 liter
Phosphate buffer
- 10.8 g K2HPO4
- 5.6 g KH2PO4
- Bring to 1 liter
Nutrient stock
- 40 g NH4Cl
- 10 g MgSO4·7H2O
- 5 g CaCl2·2H2O
- Bring to 1 liter
Trace metals
Making this is a complex proceedure so (unless you think you will be using a lot of it) there are a couple of alternatives.
- Make a soil extract by shaking a few grams of soil in water
- Use a small amount of Job's plant food spike
- Use tap water rather than distilled.
This makes a liter of full-strength, enough for 1000 L of TAP. Dilute an aliquot tenfold and use the diluted stock in making TAP
- Dissolve 50g of acid free EDTA in 250 ml of ddH2O. Heat to dissolve.
- Dissolve the following, one by one, in order, heating to approximately 100° in 550 ml ddH2O.
- 11.40 g BO3H3
- 22.00 g ZnSO4·7H2O
- 5.06 g MnCl2·4H2O
- 4.99 g FeSO4·7H2O
- 1.61 g CoCl2·6H2O
- 1.57 g CuSO4·5H2O
- 1.1 g Mo7O24(NH4)6·4H2O (Ammonium Molybdate)
- Mix the two solutions (1 & 2) together. The resulting solution should be blue-green.
- Heat to 100°C. Cool slightly, but don't let the temperature drop below 80°-90°.
- Adjust pH to 6.5 to 6.8 with 20% KOH (approximately 83 ml). Don't let the temperature drop below 70° until after the pH is adjusted.
- Make up to 1L and let stand in a 2L Erlenmeyer flask, stoppered loosly. The color should change from green to purple over a period of days.
- Remove the rust-coloured precipitate by filtering, with suction, through 3 layers of Whatman #1 filter paper in a Buchner funnel. Repeat until no more precipitate is seen on the filter paper
- Store in a brown bottle at 4°.
Links
Recipe taken from: http://www.chlamyteach.info/growthmedium.html
