2012

2012 kdorfman Mon, 08/19/2013 - 17:12

1.1: Fluorescein

1.1: Fluorescein kdorfman Mon, 11/14/2011 - 21:27

Fluorescein Absorbance and Fluorescence

Materials for 1.1

Materials for 1.1 kdorfman Mon, 11/14/2011 - 21:32

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)

Reagents for 1.1

Reagents for 1.1 kdorfman Mon, 11/14/2011 - 21:28

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


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

1.2: Fluorescein II

1.2: Fluorescein II kdorfman Mon, 11/14/2011 - 21:40

Absorbance & Fluorescence of Fluorescein, part 2

Unknowns

  • One too dilute to measure with abs (e.g., 0.0003mM; A3x10^-7M = 0.015)

  • One too concentrated to measure with Fl (e.g., above 0.01mM)

  • One too concentrated for either, e.g., 0.12 mM

Tell instructors the Abs and Fl of each, w/gain adjusted for the 1 mM solution

Give students:

  • ~6 mL 10mM NaOH

  • ~1 mL 1mM fluorescein. (Make sure Abs is correct)

Make dilution series

Check programs for correct wells.

Get a laptop for the TA to demonstrate stupid Excel tricks.

2012 unknowns

2012 unknowns kdorfman Tue, 09/18/2012 - 17:51
Unknown ~ mM ~Abs Abs blank ~ Fl Fl blank
P 0.25 5.745 0.023 38000 19
Q 0.00024 0.039 0.023 679 19
R 0.0625 4.171 0.023 32000 19