I am confused while doing the calculations. My protein concentration is 0.959 ug/uL,1.11 ug/uL and 1.5 ug/uL. I want to load 20 ug/ well and the volume that will go in each well is 30 uL. Total number of wells used is 2 for each sample which means 60 uL in total. But the total volume in the calculations that I have received is 90 uL. The loading dye being used is 15 uL (4X loading dye). For 40 ug final concentration, the volume taken is 41.71 uL(for 0.0959 ug/uL sample) and the remaining volume is the lysis buffer ( final volume=90 uL). The loading dye volume is calculated for 60 uL (1X). As for 90 uL final volume of the loading dye must be 22.5 uL. This calculation seems odd to me. Kindly help how to do the calculations to get accurate concentration of everything.
Thanks.
Ching-Tse Wu Thank you for the answer but if I want to make 10 percent extra volume. . Then can you explain the calculations?
Start with one well
The first step is to determine the volume of sample to be used for 20ug of protein. Then, use the total volume of each well to derive the amount of loading dye. Finally, subtract the sample volume and the amount of loading dye from the total volume to know how much lysis buffer to add.
Then, depending on the number of wells needed and the amount of extra volume to be retained for loading, multiply the sample volume, loading dye dosage, and lysimetric buffer. in your case it is 2(duplicate)*1.1(10% more)=2.2. Multiply all volumes by 2.2 from one well calculations.
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