When making very large dilutions (over 1000X), it is common for one to make an i

Question

When making very large dilutions (over 1000X), it is common for one to make an intermediate dilution, then to prepare the final dilution from the intermediate. To conserve reagents that are terribly expensive, such as antibodies, one usually prepares the intermediate dilution using the minimum volume of the stock that can be accurately delivered with a micropipettor (2 L if using a P20). The intermediate dilution (also known as a working stock) can then be saved and used another day.

7. Describe how to prepare exactly 1 mL of an antibody at a 110,000 dilution. You will use exactly 2 L (the minimum volume accurately measured with a P20) of the stock solution, and use phosphate-buffered saline (PBS) as your diluent.

8. Suppose you need to introduce calcium into the final antibody solution you use for labeling your cells, but do not want calcium in the intermediate dilution (working stock) because it could precipitate over time in the phosphate buffer system. A precipitate in the working stock would necessitate discarding this expensive reagent! Describe how you would prepare exactly 1 mL of a solution containing an antibody at a final dilution of 110,000 that also contains 5 mM Ca2+.

You have a 0.5 M CaCl2 stock solution available. Use PBS as your diluent. Make your

intermediate antibody dilution as in question #7.

Explanation / Answer

7.

Make three dilutions of 1/10 in the following way

2 uL P2O / ( 2uL P2O + 8 ul PBS)

Dilution = 1/10

Similarly use 2 ul from this solution 2uL / 2uL + 8 uL PBS

Dilution = 1 / 100

Again solution from this 2 uL / 2 uL + 8 uL PBS

Dilution = 1/1000

8.

Making three dilutions in a similar way where we use CaCl2 as solute and dilutent is PBS

Use 1 mL + 9 mL value

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