2023-11-23, 12 : 53 PM Quantitative Analysis Lab: Serial Dilution and Standard Curve Cons…nt Media and Control Samples Using Colorimetric Assay · Benchling Page 1 of 3 https://benchling.com/mpate445/f/lib_Z4eJ9ZSx-biology-2290g-protoco…rimetric-assay/view ? versionId=813126347&note=true&protocols=#print Quantitative Analysis Lab: Serial Dilution and Standard Curve Construction for Precise Determination of Treated Spent Media and Control Samples Using Colorimetric Assay Pupose: The purpose of this lab is to collaboratively perform a serial dilution, construct a standard curve, and employ a colorimetric assay for precise concentration determination. The focus is on analyzing treated spent media and control samples (empty vector) to enhance quantitative insights into experimental outcomes In this lab you will be performing a serial dilution in partners to create a standard curve and then use that curve to determine the concentrations of our treated spent media (experimental sample) and spent media grown with an empty vector (no insert) - this is a control. This standard curve will use a colorimetric assay to determine the concentration. 1. Specs will be turned on already so that they are warmed up. They will be set to 425 nm which is the lambda max for the colorimetric assay. 2. Begin by creating your standard curve by performing a serial dilution according to the chart below. The cholesterol standard is supplied at a concentration of 20 uM. Prepare spec tube #1 as indicated in the table. Add 3 mL of H 2 O to the remaining 4 spec tubes. Calculate what volume of Tube #1 needs to be transferred to Tube #2 etc. Tube mL H 2 O mL Standard or tube #x Concentration (uM) 1 0 6 of standard 20 2 3 of Tube 1 10 3 3 of tube 2 5 4 3 of tube 3 2 . 5 5 3 of tube 4 1 . 25 A B C D 1 2 3 4 5 6 1. Tube #5 will be the only tube with 6 mL (the rest have 3 mL). Remove 3 mL from tube #5 and discard in the waste container. THURSDAY, 2023-11-23 Table1 Explore the Notebook

2023-11-23, 12 : 53 PM Quantitative Analysis Lab: Serial Dilution and Standard Curve Cons…nt Media and Control Samples Using Colorimetric Assay · Benchling Page 2 of 3 https://benchling.com/mpate445/f/lib_Z4eJ9ZSx-biology-2290g-protoco…rimetric-assay/view ? versionId=813126347&note=true&protocols=#print 2. Add 3 mL of each spent media (both experimental and control) to separate spec tubes. 3. Add 3 mL of water to a spec tube to act as a blank 4. You should now have 8 spec tubes 5. Add 70 μL of colour reagent (labelled “CR”) to each spec tube and swirl gently to mix 6. Blank the Spec 20 by performing the following: a. With the sample holder empty, turn the left knob until the machine is "zeroed" (i.e. the needle lines up with 0% transmitted) b. Wipe the Blank with kimwipe and insert into sample holder, closing the lid c. Using the right knob, adjust the needle to 100% transmitted 7. Now measure each one of your serial dilution tubes, wiping the tube with a kimwipe before inserting in sample holder. Record the absorbance of each tube. 8. Measure the two spent media sample tubes in the same manner and record the absorbance. Microplate Reader: 1. Using the spent media samples and the standards you prepared for the Spec 20, load 100 μ L of each sample (including the blank) in triplicate in the microplate. Load the plate as indicated below. The plate will be read on a microplate reader and the values provided to you on OWL. Fill in your name(s) on the microplate template sheet and make note in your records which plate # you loaded your samples on. 2. When you are done, place the spec tubes in the bin in the sink (carefully as they are expensive!) and the serological pipettes in the washer, tips up at the front of the room. Leave all the reagents in your tube rack. 3. Results will be posted on OWL after lab. You will need to enter your concentration of glucose in the spent media samples (both control and experimental) on a Google sheets to collect class data. More info will come in an announcement.