Follow-up to the September 27 Webinar

Submitted by Toby Horn on 30 September, 2012.

Resources:

The Department of Education's OVAE has supported development of www.stemtransitions.org

Led by CORD, this effort by more than 30 community college faculty from around the US has produced (now 61) lessons/activities that live in career clusters. Biotech, being the interdisciplinary/cross-applicable pursuit, encompasses activities that you can find under Agriculture, Food and Natural Resources, Health Science, STEM (and maybe even Manufacturing).

You have to register, but it's free.

The lesson packages have background for faculty, lesson plans, assessments, etc.

Pedagogy:

Enabling the Synergy of Teamwork. Even simple methods, such as a protein assay, can be adjusted to ensure that in teams, every member pipettes, every member records, every member checks and supports their 'mate's technique to ensure GLP (in a rotating fashion).

For example, we conduct an eyeball protein assay for starters given that students need to work through (for them) a great deal of math "Math of Milk." Someone else has now produced "Got Milk?" for Bio-Rad. Here's how we've done this since 1986.

Students work in teams of 3.

They learn to pipette with rubber bulbs (WORST CASE SCENARIO - and because we could't afford the pi-pumps to start) e.g. https://www.labdepotinc.com/Product_Details~id~344~pid~12713.aspx (the 35 ml size) using PRE-MED (write me for instructions).

Students then read the label on a milk carton and calculate the SI concentration of protein (mg/ml).

+++After each boardwork segment, what was written gets erased! so students have to rely on what they wrote in their lab books.

Students next read the Bio-Rad microassay method and discover that the range indicated is ug/ml!

We work thru a dilution of mg/ml to get ug/ml (and instruct students to get actually to 10s of ug/ml) so they will be able to add 0.1 to 0.2 ml of sample for each assay. One member adds water, next member adds sample, third member adds reagent = every team member pipettes.

We use 24-well dishes - over and over and over. They work great! and can be dipped in 10% bleach plus detergent and then rinsed several times under running water, minimizing discoloration of the plastic. The Coomassie blue dye reacts with the plastic!

For HS students, the first run is called the "Goldilocks Assay" too much, too little, just right (works fine for HS to use this terminology) Could also be a "cereal dilution" awfully punny for older students. As a team, the students conduct a serial dilution where each member adds 0.9 ml of water to 2 places in a diluent row, then, they start by the first student making the 1:10 dilution (from the milk sample), handing the milk pipette to the next student who makes the 1:10 of the first dilution to 1:100 and the third student gets the pipette to make the 1:10 to get 1:1000. This way, they've embodied the serial dilution process one-by-one-by-one.

NEXT they run the dilutions as samples, adding 0.1 ml of each sample to 0.7 ml of water (ie person 1 adds 0.7 ml of water to each of several tubes. Person 2 adds 0.1 ml of each dilution and person 3 adds the reagent, and the other 2 mix as person 3 adds the reagent. (The reagent settles to the bottom if not mixed right away. Toothpicks are great for mixing.)

Instructor points out the reasonable color (which should be the 1:100 dilution).

Next, the students prepare an assay where all the samples "should" be in the range and figure out how to set up assay tubes to see colors from brown (0 protein added) to 25 ug.

(Calculate 0.2 ml reagent for every ml (another reason why the 24-well plates are great--1 ml is only 1/3 the well capacity) and 0.8 ml for water plus sample (1:100 dilution) guide students to add 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 ml of 1:100 dilution of milk (beforehand adding the volume of water to yield 0.8 ml total with sample). Final addition is the reagent even tho it's the first to consider. Students will get colors ranging from brown to royal blue. (Indication of dose-response)

++I prefer the Bio-Rad reagent vs Pierce because the Bio-Rad reagent 0 protein color is a different color to the colors with any protein added.

If the students conduct the assay in duplicate, you can force them to make a judgment call about colors (how similar are the duplicates?).

The final run can be to combine the milk assay with the real standard (BSA--5 mg/ml diluted to 50 ug/ml) and run all of them thru a spec.

  • Pipetting technique
  • Math
  • Calculations
  • Dilutions/Sampling
  • Operations
  • Developing an eye for "What IT Should Look Like."

Lookfors include:

same level (=same volume) for completed assay in each well.

no drops of dye along the rim (all reagent mixed in)

duplicates same color

Goldilocks goes from opaque bright blue (too much) to nearly brown (too little)

Assay of 1:100 dilution goes from brown thru slate blue to dark clear blue to bright blue

Spec assay with BSA looks like the Bio-Rad microassay (note 0 protein is not 0 absorbance)

You can reuse the pipettes. We pull the cotton out and keep some dishes, 1 ml pipettes and red bulbs in a box w/ premed for students to practice any time (PRACTICE PIPETTING)

You can reuse the 24-well dishes

You can have a rodeo by team and then by individual (providing a starting milk sample that's actually diluted a bit to dfferent extents eg 1:1.35 (add 0.35 ml water to 1 ml of milk) 1:1.6, etc.)

With 9th graders and 90 min lab periods, we did "pre-lab, intro piptting, technology of tubes to 24-well dishes--importance of molding in the well identifiers) and reading the label. Goldilocks Day 2, the in-range dilution Day 3, Eyeball assay with standard Day 4. For day 5, students brought in samples of other liquids or we made cheeses and they examined the milk vs whey. (We had no access to spectrophotometers.). In a 3h lab with older students (e.g., CC, you could run the Goldilocks Assay (incl practice pipetting and calculating) and then the microassay with standard if you find students are comfortable enough w/ the math. If not, run the Goldilocks and the 1:100 dose-dependent colors and next lab period run the standard and diluted milk in different doses (not the rodeo conditions).

Visit my (OLD) website for the protein assay at https://carnegiescience.edu/first_light_case/horn/PROTsite/proteinindex.html