BISC300 Lecture 9: Lecture 9 Dilutions Practice

Standard Plate Count Assessment

You want to make lemonade from scratch. This means combining lemon juice from lemons, sugar and water. This is really a dilution problem like the protocol described. The lemon juice is 2 times more concentrated than you want it to be. How would you dilute the lemon juice accordingly? Your answer must include the volumes of lemon juice and water (diluent) that need to be mixed together and also what the total volume will be after the dilution.

1:5 dilution = 1/5 dilution = 1 part sample and 4 parts diluent in a total of 5 parts. If you need 10 ml, final volume, then you need 1/5 of 10 ml = 2 ml sample. To bring this 2 ml sample up to a total volume of 10 ml, you must add 10 ml - 2 ml = 8 ml diluent.

A. Your friend wants to perform 1/10 and 1/100 dilutions of the concentrated lemon juice. The person does not understand what this means and asks you for help. How would you explain to her what this means? How should she perform these dilutions?

1/10: _____ ml lemon juice and ______ ml water

1/100: _____ ml lemon juice and ______ ml water

A. You take 1 mL of concentrate juice and add 9 mL of water. What dilution did you make here?

B. You take 10 mL of concentrated juice and add 90 mL of water.What dilution did you make here?

C. Were your answers in A and B the same or different? Explain your answer.

Please answer all the questions answering only one question is not helpful :

Calculations on Multiple Stock Solutions—be sure to include the amount of water added and include all units

11. Calculate how to use the 1 M Tris and 0.2 M EDTA stock solutions to make the 400 ml of a of 10 mM Tris and 1 mM EDTA solution.

12. Calculate how to use a 3 M NaCl and 10% NP40 stock solutions to make a 500 ml of a 300 mM NaCl and a 0.2% NP40 working solution.

Calculations on Simple Dilutions—be sure to include the amount of water added and include all units

13. Calculate the volumes necessary to make 200 ml of a 1:5 dilution of a sample.

14. Calculate the volumes necessary to make 50 ml of a 1:8 dilution of a sample

15. Calculate how you would dilute a cell suspension containing 3,000,000 cells/ml to obtain 10 ml with 100,000 cells/ml

16. Calculate the volume of a cell suspension containing 800,000 cells/ml you would need to obtain a total of 250,000 cells

Practice Calculations on Serial Dilutions—be sure to include the amount of water added and include all units

17. Calculate and describe (or diagram) how you would use a 10% SDS solution to prepare a final volume of 20 ml of each of the following solutions: 8%, 4%, 2%, 1% 0.5%.

18. Calculate and describe (or diagram) how you would use a 5 M Tris solution to prepare 200 ml of each of the following solutions: 1M, 100 mM ,10 mM , 1 mM, 0.1 mM.

How much of the first dilution should you make to perform the following dilutions?

19. 300 ml of a 1:5 dilution

20. 100 ml of a 1:10 dilution

21. What is the minimum volume you should pipette with the following? (include units with your answers):

P2 P10 P20 P100 P200 P1000

22. How accurate was your pipetting?

23. Was it better with the larger volume or smaller volume pipettor? Provide rationale.

(If there was a consistent problem with one pipettor please tell the instructor.)

Standard Curves

24. Complete a standard curve for the data above and tape a printed version into your notebook.

25. What is the R-squared value of your trend line? Is this good or bad… i.e. would you repeat the assay?

26. What is the protein concentration of (Explain/show your calculations.)

Unknown #1 = Unknown #2 =

Data for DNA Concentration Procedure

Attach a labeled printout of the plate reader raw data as well as your generated standard curve to this lab.

26. What is the concentration of your unknown DNA sample?

27. What is the R ² for your standard curve data? Why is it important that the R² is >0.95?

28. Comment on the purity of your DNA... discuss- How can you determine the purity of DNA? What are common contaminants of DNA purified from cells?

please answer all the question answering only one question is not helpful .
Practice Calculations on Serial Dilutions—be sure to include the amount of water added and include all units

17. Calculate and describe (or diagram) how you would use a 10% SDS solution to prepare a final volume of 20 ml of each of the following solutions: 8%, 4%, 2%, 1% 0.5%.

18. Calculate and describe (or diagram) how you would use a 5 M Tris solution to prepare 200 ml of each of the following solutions: 1M, 100 mM ,10 mM , 1 mM, 0.1 mM.

How much of the first dilution should you make to perform the following dilutions?

19. 300 ml of a 1:5 dilution

20. 100 ml of a 1:10 dilution

21. What is the minimum volume you should pipette with the following? (include units with your answers):

P2 P10 P20 P100 P200 P1000

22. How accurate was your pipetting?

23. Was it better with the larger volume or smaller volume pipettor? Provide rationale.

(If there was a consistent problem with one pipettor please tell the instructor.)

Standard Curves

24. Complete a standard curve for the data above and tape a printed version into your notebook.

25. What is the R-squared value of your trend line? Is this good or bad… i.e. would you repeat the assay?

26. What is the protein concentration of (Explain/show your calculations.)

Unknown #1 = Unknown #2 =

Data for DNA Concentration Procedure

Attach a labeled printout of the plate reader raw data as well as your generated standard curve to this lab.

26. What is the concentration of your unknown DNA sample?

27. What is the R ² for your standard curve data? Why is it important that the R² is >0.95?

28. Comment on the purity of your DNA... discuss- How can you determine the purity of DNA? What are common contaminants of DNA purified from cells?