Concept #4: When making very large dilutions (over 1,000X), it is common for one

Question

Concept #4: When making very large dilutions (over 1,000X), it is common for one to make an intermediate dilution, then to prepare the final dilution from the intermediate. To conserve reagents (especially expensive ones!), one usually prepares the intermediate dilution using the minimum volume of the stock that can be accurately delivered with a micropipettor (2 uL if The intermediate dilution (also known as a working stock) can then be saved and used for another application (critical when working with expensive materials!) using a P20). 7. Describe how to prepare exactly 0.5 mL of antibody at a l 10,000 dilution. Use only 2 of the stock solution, and use phosphate-buffered saline (PBS) as your diluent. 8. Suppose your current experiment requires using a mild detergent during your antibody incubation step, but you do not want to add it to the intermediate dilution (working stock) because it would interfere with cell function in the next experiment for which you plan to use the working stock (remember, these are expensive reagents!). Describe how you would prepare exactly 1 mL ofantibody at a final dilution of I-.5,000 in a solution containing 0.1% v/v) detergent. You have a stock solution of 10% (v/v) detergent and the intermediate antibody dilution you described in question #7. Use PBS as your diluent.

Explanation / Answer

7. To dilute 2ul into 10,000x, we need 20,000L = 20L of PBS, which is not feasible in routine labs. Thus, we need to first make an intermediate stock and further dilute this stock to prepare working stock. The feasible dilutions could be 10x and 1000x (10x dilution and then 1000x dilution), 100x and 100x, 2000 and 10x and so on. Of these, 2000x and 10x one is the most convenient. For this, we need to mix 2ul of antibody into 2000ul of PBS (=1000x dilution). This is intermediate stock. Note that a 10x further dilution will make the desired 10000x dilution. Thus, this intermediate tube is 10X concentrated. To prepare 500ul (=0.5mL), we need to add 500/10 = 50ul of the intermediate stock and mix with 450 ul of PBS.

8. The previous intermediate stock is of 1000x dilution. Another 5x dilution will make it 5,000x dilution. To prepare 1000ul (=1mL), we need to add 1000/5 = 200ul of the intermediate stock.

100x dilution of 10% detergent will make 0.1%. Since the final volume is 1000ml, the volume of the detergent needed is 1000/100=10ul

Thus, we need 200ul of the intermediate stock and add 10ul of the detergent stock and mix with 1000-210=790ul PBS

Related Questions

Navigate

• Browse (All)
• Browse C
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.