Yaamini’s Notebook: DNR BCA Assay Round 3

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Once again, Jose and I are using the protocol generated in December to quantify the protein concentrations my samples and Laura’s samples. We’ll also quantify the protein in Rhonda’s larval geoduck samples. It’s a lot to keep track of! The end goal is to obtain the µL of protein required for 100 µg of protein in our samples, and the corresponding amount of 50 mM NH4HCO3 in 6M urea.

Step 1: Prepare reagents for 90-100 samples

Step 2: Plan microplate arrangement

Figure 1. Microplate arrangement

Step 3: Pipet 10 µL of either standard or sample into the corresponding microplate well

Step 4: Prepare BCA working reagent

The BCA working reagent should only be prepared right before plate incubation and reading.

  • ((8 standards x 5 plates) + 100 samples) x (3 replicates each) x (200 µl of working reagent per well) = 104,000 µL working reagent = 104 mL working reagent
  • Used 110 mL Reagent A and 220 µL Reagent B to make a 50:1 Reagent A to B ratio.

Step 5: Added BCA working reagent to each well

Figure 2. Microplate 1

Figure 3. Microplate 2

Figure 4. Microplate 3

Figure 5. Microplate 4

Figure 6. Microplate 5

Step 6: Used Genome Sciences incubator and plate reader

Step 7: Calculated volume of protein needed for 100 µg per sample

from the responsible grad student http://ift.tt/2ri73r2
via IFTTT

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