Download IndiAgeBasicAssay

IB Biology / IHS / Rev 16-17
Way to Go, Indigo! The Basic Assay
Adapted from Diane Sweeney, “Way to Go, Indigo!” Exploring Life Lab Manual © 2002
Special thanks to Mr. Art Roberson, Technical Sales Manager at Genencor International,
for donating IndiAge Super GX Plus enzyme and providing technical assistance!
Materials
Equipment
Reagents & consumables
Part 1.
Part 1.
Sharpie™ marker
two 250-mL beakers
two graduated cylinders
two forceps
PPE: goggles, aprons
Part 2. two plastic wash basins and 400-mL beakers
two scrubbers
PPE: goggles, aprons
magnifier or dissecting microscope
fabric cutting station: dark denim fabric & scissors
IndiAge solution (2% IndiAge in pH 5 buffer)
pH 5 buffer
Ziploc™ bags
label tape
envelope
Part 2. paper towels
PPE: nitrile gloves
55oC water (~1200 mL/group), heated in water bath
Part 1.
1.
Cut a 3 in. x 9 in.* piece of dark denim. Then, cut it into thirds, giving you three 3 in. x 3 in. swatches.
2.
To distinguish the experimental denim swatch (the one you will treat with IndiAge), cut off one of its
corners. Leave a 2nd swatch (it will be Control 1) uncut. Write “C2” (for Control 2) on the back of the
3rd denim swatch with a Sharpie™ and save it in an envelope labeled with your team letter until Day 2.
3.
Label one beaker or large cup, “Buffer with IndiAge.” Label a second beaker or cup, “Buffer Only.”
4.
In each beaker stand up a small Ziploc™ bag. Pour 200 mL of buffer with IndiAge solution into the bag
in the “Buffer with IndiAge” beaker, and 200 mL of pH 5 buffer into the “Buffer Only” bag.
5.
Using forceps, lower the denim swatch with the cut corner into the “Buffer with IndiAge” bag. Lower
the Control 1 swatch in the beaker labeled “Buffer Only.” Push air out and zip both bags closed.
6.
Allow the two denim swatches to incubate at room temperature for 3-4 days.
Part 2.
7.
Remove both swatches from their beakers. Keeping them SEPARATE, rinse the swatches WELL under
HOT running water. NOTE: The baggies’ liquids must be neutralized with baking soda before disposal.
In denim processing facilities, jeans are placed in industrial washers to break the hydrolyzed cellulose fibers and
release indigo molecules. We’ll hand wash our swatches with scrubbers to simulate this process!
8.
Put on nitrile gloves so your hands don’t turn blue. 
9.
Put the denim swatches in separate basins with 400 mL hot (55oC) water each. Scrub each of the
swatches with a scrubber underneath the water. Lift them out and squeeze repeatedly. Continue
scrubbing the swatches for 12 full minutes, changing the water every 4 min. Try to give both swatches
the same all-over scrubbing and squeezing treatment.
10.
Wring out the swatches one last time and lay them flat on layers of paper towel to dry overnight.
11.
Compare all three denim swatches when dry. If time permits, use a magnifying glass or stereoscope to
look for differences in the fibers. What macroscopic or microscopic effect(s) did IndiAge have?