1 fluorescein

1 fluorescein kdorfman Mon, 08/19/2013 - 17:09

1.1

1.1 kdorfman Mon, 08/19/2013 - 17:14

Materials

  • one balance per table (borrow from Bill Schmitt)
  • weighboats
  • non-sterile tips
  • Mats
  • Gloves
  • small glass culture tubes (~15/pair)
  • rack for tubes

  • beaker for water

  • 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@ISB)

10mM NaOH

MW = ~40

10mM = 0.01M = 0.4g/L

To make M NaOH/
with g NaOH pellets
use L H2O

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)

1.2

1.2 kdorfman Mon, 08/19/2013 - 17:18

Absorbance of Fluorescein

Per pair:

  • Black 96 well plate (not necessarily sterile, but clean)
  • 1 mM fluorescein (~ 1 mL)
  • 10 mM NaOH (~ 4.5 mL)
  • 2 2-mL tubes to make 2 mL of 10µM fluorescein (each partner does one)

Computers and Plate Readers

  • Check program Repeatability (Abs 485 nm; layout A1-B12, volume 150 µL)
  • Check program FL-Abs-by-2 (Abs 485 nm; layout C1-2, D1-12, volume 150 µL)
  • 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)

solutions

solutions kdorfman Fri, 09/06/2013 - 18:47

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)

10mM NaOH

MW = ~40

10mM = 0.01M = 0.4g/L

1.3

1.3 kdorfman Mon, 08/19/2013 - 17:18

Corrections

Read the plate only once

Pipet 150 µL of each unknown in E3 - 12 as needed

Programs

  • Serial2&5_Abs
  • Serial2&5_FL
  • E1 &E2: blank E3-E12 for unknowns
  • F1 - F12: dilute by 2
  • G1 - G12: dilute by 5

Materials:

  • ~1 mL 1mM fluorescein (try to make it right via Abs)
    • E = 50358/M
    • A = 50358/M * C
    • at 10 µM, A = 0.5038
    • dilute 1 mM 1/100 to make 10 µM, take Abs
  • 5 mL 10 mM NaOH
  • black plate from last time (or new one if old one has no clean wells)

Conversion factors

Abs = 59.7*(mM)-0.341 up to mM=0.03125

FL = 10^6 * mM +173.06 up to mM = 0.008

FL = 2 X 10^6 * mM + 19.624 up to mM = 0.00195

Unknowns

  • Make dilution series from the "1mM" stock.
  • find lowest useful concentration for Abs. Dilute slightly for unknown 1 - must be done with FL. Abs is bad below 0.0005 and meaningless below 5 x 10^-7mM
  • find a concentration too high for fluorescence, but OK for abs, e.g., 0.05.
  • find a concentration too high for either. must be >0.125

2013:

label ~mM Abs FL
P 0.0001 ~0 210
Q 0.015 0.8699 16496
R 0.1 4.955 26780
test-fluorescein_ps1.xlsx

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

fluorescein_info_sheet.pdf

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