04 - Put the M into STEM: Quantitative Techniques for Biotechnology
Step 2: Performing the Quantitative ELISA
In this simulation, we have combined the antigen, primary antibody, and secondary anti- body steps. You will be testing two samples: control (C) and experimental (E).
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1. ORIENTATE your strip so that the longer box of wells is on the left. 2. LABEL the left wells 1 through 4. LABEL well 5 as “C” and well 6 as “E”. 3. PIPET the provided standard curve. a. b. With a new pipet tip, PIPET 100 µL of 1:4 solution to well 2. c. With a new pipet tip, PIPET 100 µL of 1:8 solution to well 3. d. With a new pipet tip, PIPET 100 µL of 0 solution to well 4. 4. PIPET the simulated patient samples. a. With a new pipet tip, ADD 100 µL of Control sample to well 5 b. With a new pipet tip, ADD 100 µL of E sample to well 6. 5. INCUBATE for 5 minutes at room temperature. 6. INVERT the strips over the sink or a stack of paper towels to remove the samples. Gently TAP the strips 4-5 times onto a fresh paper towel. DISCARD the wet paper towels. 7. Using a transfer pipet, ADD PBS buffer to each well until it is almost full – around 9 drops. 8. INVERT the strips over the sink or a stack of paper towels to remove the samples. Gently TAP the strips 4-5 times onto a fresh paper towel. DISCARD the wet paper towels. 9. Using a new transfer pipet, ADD 100 µL (2 drops) of substrate solution to all wells. 10. INCUBATE for 5 minutes at room temperature. 11. ANALYZE the strip. PIPET 100 µL of Ab solution to well 1 (provided in a starting concentration of 20 µg/mL).
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