Materials

• 1 black-walled plate per pair
• non-sterile tips
• Mats
• Gloves
• Serial dilution by 5 for demonstration purposes.
  • First tube saturated
  • Last tube blank.
• blue flashlights & yellow filters

Computers and Plate Readers

• Check program Repeatability (Abs 485 nm; layout A1-B12)
• Wahoo folders must be named Tues01, Tues02, ...Tues12 and Thur01, Thur02 ... Thur12, even though the user names in the Optima program are Tue01 and Thu01, etc.
• Import program to groups
• Check network connections (in “my computer” connect the Z drive to Wahoo as kdorfman, kd@hh3)

10mM NaOH

MW = ~40

10mM = 0.01M = 0.4g/L

Need:
- 25 tubes of 10 mL each = 250 mL NaOH

• 13 tubes of 2 mL each = 26 mL fluorescein A

• 13 tubes of 2 mL each = 26 mL fluorescein B

= total of 300 mL NaOH needed

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1 mM Fluorescein

in 10 mM NaOH

It fades with time, so check the concentration of the old stocks.

MW = 332

1mM = 0.332g/L = 0.033g/100 mL

A = Ecl

Molar extinction coefficient of fluorescein at 485nm = 50358/mol.cm

so Absorbance 10^-5M = 0.50358

(1/100 of a 1mM solution =10^-5M)

Make A & B slightly different.

(Different enough so that they are different by the t-test. Make one above and one below the “actual” value.)

Be sure to measure a blank. In 2012, the blank was ~0.25, so Abs readings should be ~0.75