Potato Germplasm Enhancement Laboratory

Obihiro Univerisity of Agriculture and Veterinary Medicine
West 2-11, Inada, Obihiro, Hokkaido 080-8555, Japan

Recipes for solutions and chemicals

1. Measure 800 ml of Ethanol in a 1000 ml graduated cylinder.
2. Measure 100 ml of 10 mol/l Ammonium acetate solution (Nakaraitesk 024-32), and add into the graduated cylinder containing ethanol (1).
3. Measure 33.3 ml of distilled water in a graduated cylinder used in (2), and add.
4. Place a stir bar and mix on a magnetic stirrer.
5. Dispense into two 500 ml dark bottles and store in a freezer.
   

1. Add about 400 ml of distilled water in a 1000 ml beaker.  With stirring on a magnetic stirrer, add gradually 10 g of CTAB (Hexadecyltrimethyl ammonium bromide, C19H42NBr).
2. Add 40.91 g of Sodium chloride, and then, 5 g of Polyvinylpyrrolidone (PVP-40).
3. Add 25 ml of 2 M Tris-Cl buffer (pH 8.0) using a 10 ml pipette.
4. Add 20 ml of 0.5 M EDTA (pH 8.0) using another 10 ml pipette.
5. Fill with distilled water up to approximately 500 ml and warm (up to the temperature you feel warm) to dissolve completely.
6. Transfer to a 500 ml graduated cylinder and adjust the volume to 500 ml.
7. Transfer back to the 1000 ml beaker and stir well.
8. Dispense 100 ml each into a 100 ml bottle, and autoclave.
9. Before use of the bottle, add 200 μl of 2-mercaptoethanol.

1. Dissolve all components in 30 ml of distilled water in a 100 ml Erlenmeyer flask.
2. After completely dissolved, fill up to 50 ml with sterile water
3. Dispense 1 ml each into 1.5 ml tubes.

1. Thaw frozen Formamide on a bench.
2. Place 9.8 ml of Formamide in a 100 ml beaker, and add 0.2 ml of 0.5 M EDTA (pH8.0).
3. Add 5 mg of Bromophenol blue and 5 mg of Xylene cyanol, and mix completely using a stirrer.
4. Dispense 1 ml each into 1.5 ml tubes.

1. Dissolve 100 mg of gelatin in 10 ml of distilled water in a 100 ml Erlenmeyer flask by heating.
2. Autoclave.
3. After cooled, dispense 1 ml each into 1.5 ml tubes.

1. Add distilled water to slightly below the line of 1000 ml in the 1000 ml bottle labeled as “0.25 M HCl for depurination”.
2. Measure 21.6 ml of Hydrochloric acid with a 10 ml pipette, and add to the bottle.
3. Fill up to 1000 ml with distilled water.
4. Mix by shaking and rotating the bottle.
   

1. Transfer all contents of one bottle “Gold hybridization buffer” contained in an ECL kit into a 1000 ml beaker (exactly 500 ml are contained).
2. With vigorously stirring on a magnetic stirrer, add gradually 25 g of Blocking reagent.
3. Add 14.61 g of Sodium chloride.
4. Warm the solution (up to the temperature you feel warm), and continue to stir for 2-3 hours.
5. Dispense into 50 ml Falcon tubes and store in a freezer.
   

1. Measure 29.22 g of Sodium chloride in a 200 ml beaker.  Fill with distilled water to approximately 100 ml, and dissolve completely on a magnetic stirrer.
2. Transfer to a 100 ml graduated cylinder and adjust the volume to 100 ml with distilled water.
3. Transfer to a 100 ml bottle and autoclave.

1. Add distilled water to slightly below the line of 1000 ml in the 1000 ml bottle labeled as “0.4 M NaOH for alkaline blotting”.
2. Measure approximately 16 g of Sodium hydroxide, and add into the bottle with stirring on a magnetic stirrer (a stir bar sitting in the bottle).
3. After dissolved completely, fill up to 1000 ml with distilled water.
   

1. Add 390 μl of 10x dye and 510μl of sterile water in a 1.5 ml tube, and mix well.

1. Add distilled water to about half volume in a 5000 ml plastic beaker.
2. Add gradually 20 g of SDS (Sodium laurylsulfate or Sodium dodecyl sulfate).
3. Add gradually 1800 g of Urea (measure 200 g of Urea nine times in a disposable plastic beaker, and add).
4. Measure 125 ml of 20x SSC in a 250 ml graduated cylinder, and add.
5. Fill with distilled water up to approximately 5000 ml.  Dissolve all components completely (it may take more than one hour).
6. After completely dissolved, transfer to a 5000 ml plastic graduated cylinder and adjust the volume to 5000 ml.
7. Transfer to a 10 liter tank.
   

1. Mix all components in a 200 ml Erlenmeyer flask and autoclave.
2. After cooled, dispense 1 ml each into 1.5 ml tubes.

1. Measure 500 ml of 20x SSC in a 500 ml graduated cylinder, and add into a 10 liter tank.
2. Measure 4500 ml of distilled water in a 5000 ml plastic graduated cylinder, and add into the 10 liter tank.
3. Mix the solutions on a magnetic stirrer (a stir bar always sitting in a tank).
   

1. Add 3000-4000 ml of distilled water in a 5000 ml plastic beaker.
2. Measure 876.6 g of Sodium chloride (use a plastic disposable beaker), and add gradually to dissolve by stirring.
3. Measure 441.15 g of tri-Sodium citrate dihydrate (use a plastic disposable beaker), and add gradually to dissolve by stirring.
4. Fill the volume up to approximately 5000 ml with distilled water.
5. After dissolved completely, adjust pH to 7.0 with drops of Hydrochloric acid (maybe a few drops) by monitoring with a pH meter.
6. Transfer to a 5000 ml plastic graduated cylinder and adjust the volume to 5000 ml.
7. Transfer back to the 5000 ml plastic beaker and readjust pH to 7.0.
8. Dispense into 1000 ml bottles and autoclave.
   

1. Measure 1000 ml of 20x SSC in a 1000 ml graduated cylinder, and add into a 10 liter tank.
2. Measure 3000 ml of distilled water in a 5000 ml plastic graduated cylinder, and add into the 10 liter tank.
3. Mix the solutions on a magnetic stirrer (a stir bar always sitting in a tank).
   

1. Add 500-600 ml of distilled water in a 1000 ml beaker.  With stirring, add and dissolve 242.28g of Tris (hydroxymethyl) aminomethane.
2. Measure visually 57.1 ml of Acetic acid in a 100 ml graduated cylinder, and add.
3. Measure 100 ml of 0.5 M EDTA (pH8.0) in a 100 ml graduated cylinder, and add.
4. Fill with distilled water up to approximately 1000 ml.  After Tris is dissolved completely, all are decanted into a 1000 ml graduated cylinder and adjust to 1000 ml with distilled water.
5. Transfer to a 1000 ml bottle and autoclave.
   

1. Add approximately 800 ml of distilled water in a 1000 ml beaker.  With stirring, add and dissolve 108 g of Tris (hydroxymethyl) aminomethane and 55 g of Boric acid.
2. Add 40 ml of 0.5 M EDTA (pH8.0) using a 10 ml measuring pipet.
3. Fill with distilled water up to approximately 1000 ml.  After Tris is dissolved completely, all are decanted into a 1000 ml graduated cylinder and adjust to 1000 ml with distilled water.
4. Transfer to a 1000 ml bottle and autoclave.
   

1. Place 500 μl of 2M Tris-Cl buffer (pH 8.0) and 200 μl of 0.5M EDTA (pH 8.0) in a 100 ml bottle.
2. Add 99.3 ml of distilled water.
3. Autoclave.

1. Place 100 μl of 10x dye and 700μl of TE buffer in a 1.5 ml tube, and mix well.