Solution Preparation: x1 PBS, 20 µl Methylene-Blue, 1 mM MCH

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Necessary Materials

1. x10 Phosphate-Buffered Saline (PBS) stock solution

  • Corning: Composition of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 KH2PO4 with a pH of 7.4

2. Methylene Blue Powder

  • MW = 319.85 [g/mol]
  • Product Number: M9140-25G

3. 6-Mercapto-1-hexanol (MCH)

  • MW = 134.24 [g/mol]

4. 25 mM Phosphate Buffer (PB)

5. Nuclease Free Water

  • Ambion Life Technologies Corporation, Product Number: AM9939

Procedure

x1 PBS Solution, V = 250 ml

1. Use formula: C1 * V1 = C2 * V2 to find volume of x10 PBS stock solution that needs to be diluted to make x1 PBS solution.

  • C1 = x10, V1 = x
  • C2 = x1, V2 = 250 ml

2. Using above information, V1 =25 ml of x10 PBS will be diluted.

3. Mix 25 ml of x10 PBS with 225 ml of nuclease free water.

4. Label: x1 PBS in Nuclease Free Water (Initials, Date).

5. Store at RT.



20 µM Methylene-Blue Solution, V = 10 ml

1. Goal: Powder MB → 20 mM MB → 20 µM MB

  • Note, most of the time dilution of 20 mM MB → 20 µM MB is the only step that will be necessary, because stock 20 mM MB solution can last for a long time.


2. Powder MB → 20 mM MB

2a. V = 5 ml

2b. MW of powder MB = 319.85 g/mol

2c. To make 5 ml of 20 mM MB: 31.985 mg of powder MB + 5 ml of 25 mM PB

2d. Vortex and label properly.

2e. Store in the -4°C fridge.


3. 20 mM MB → 20 µM MB

  • IMPORTANT: This dilution has to be done in x1 PBS, because ions in PBS help in screening the charge between DNA strands!!!

3a. Use formula: C1 * V1 = C2 * V2 to find volume of 20 mM MB stock solution that needs to be diluted to make 20 µM MB solution.

  • C1 = 20 mM, V1 = x
  • C2 = 20 µM, V2 = 10 ml

3b. Using above information, V1 = 10 µl of 20 mM MB will be diluted.

3c. Mix: 10 µl of 20 mM MB + 10 ml of x1 PBS.

3d. Vortex and label properly.

3e. Store in the -4°C fridge.

1 mM MCH Solution, V = 5 ml

1. Goal: Always make fresh 1 mM MCH!

2. Stock MCH → 1 mM MCH

2a. Vfinal = 5 ml

2b. C of stock MCH = 0.0073 mol/ml

2c. To make 5 ml of 1 mM MCH: 1 μl of stock MCH + 5 ml of 25 mM PB

2d. Vortex and label properly.