2012
2012 kdorfman Mon, 08/19/2013 - 17:121.1: Fluorescein
1.1: Fluorescein kdorfman Mon, 11/14/2011 - 21:27Fluorescein Absorbance and Fluorescence
Materials for 1.1
Materials for 1.1 kdorfman Mon, 11/14/2011 - 21:32Materials
- 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 - 21:2810mM 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 - 21:40Absorbance & 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 - 17:51Unknown | ~ 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 |