Tip: for a 4-fold dilution, pick a base volume that can be divided by 3, and a t

Question

Tip: for a 4-fold dilution, pick a base volume that can be divided by 3, and a transfer volume = the base volume divided by 3.

What would you use as base volume and transfer volume?

You to prepare a four-fold substrate dilution series in 8 steps ranging from is 30 mM. 1.8 uM final concentration in quadruplicate. Your highest FINAL substrate concentration is 30mM. In your final assay plate, you are mixing 100 ul of substrate solution, 50 ul of inhibitor solution, and 50 ul of enzyme solution. Your highest INTERMEDIATE substrate concentration therefore needs to be what? Of each substrate solution you need 100 ul well your final assay plate, you are running 4 replicas, therefore you need at least 400 ul per in some extra of each substrate solution. You need to add some extra volume to make up for loss during pipetting, since we are using a 4-fold let's pick a base volume that can be divided by 3. For a 4-fold dilution series, the volume to be transferred during each step is the base volume divided by 3. What would you use as base volume and transfer volume? Draw a diagram explaining how you would generate this dilution series (S1-S8), indicating the concentration of each, the base volume, and the transfer volume:

Explanation / Answer

the base volume would be 400uL

400uL of 30mM substrate solution, transfer 100uL of this solution and dilute to 400uL (7.5mM).

Transfer 100uL from the dilution and dilute to 400uL (1.875mM).

Transfer 100uL from the dilution and dilute to 400uL (468uM)

Transfer 100uL from the dilution and dilute to 400uL (117uM)

Transfer 100uL from the dilution and dilute to 400uL (29.25uM)

Transfer 100uL from the dilution and dilute to 400uL (7.3125uM)

Transfer 100uL from the dilution and dilute to 400uL (1.82uM)

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