The composition of solutions as reported in scientific journals is the final con

Question

The composition of solutions as reported in scientific journals is the final concentrations of components and the pH only. If you wish to prepare the solution in your own lab, you need to do the calculations necessary to create a “recipe.”

3. An experiment you wish to reproduce lists one of the buffers used as “Tris-buffered saline (TBS, 150 mM NaCl, 20 mM Tris, pH 7.5).” Describe how to prepare 500 mL of TBS, pH 7.5. The formula weights of NaCl and Tris are 58.44 and 121.14, respectively. Note that solutions of Tris have a very basic pH (~11) and may require significant volumes of HCl to adjust the pH to physiologic levels. You need to describe how to allow for that added volume when you writeyour “recipe.”

4. You decided to prepare the TBS described above as a 10X stock solution that can be diluted with ddH2O to a 1X solution “on demand.” Describe how to prepare 200 mL of 10X TBS. Report the concentration of the individual components in the 10X stock, the final pH, how much of both of the solids to combine, and how to allow for the volume required to adjust the pH.

When planning an assay, one often needs to determine how to deliver a given mass of protein or a certain number of cells in a set volume of solution. This requires calculating backwards to determine what concentration (e.g., mg protein/mL, number cells/mL) will meet the requirements of the assay.

5. You need to deliver 250 ng of protein per well to a microtiter plate in a volume of 100 L per well. To what final concentration (per mL) should you dilute your protein solution?

6. You have a pellet containing 2.8 x 106 cells in the bottom of a centrifuge tube. If you need to deliver 0.5 x 106 cells in a volume of 50 L per tube to a series of assay tubes, what volume of

PBS should you use to resuspend the cell pellet? (Hint: first, determine the concentration per mL that will deliver 0.5 x 106 cells in 50 L.)

Explanation / Answer

3) To make 1M solution(or 1000mM ) -58.44g of NaCl is used in 1 litre.

So to make 500 ml 29.22 g of NaCl is required.

to make 1000mM in 500 ml NaCl required is 29.22g

so for 1 mM =29.22/1000

Therefore to make 150 mM 29.22/1000X150=4.38g of NaCl is required.

Tris-Hcl

To make 1000mM of Tris dissolve 121.14 g in 1 litre.

for 500 ml (1000mM) its 60.57g Tris.

for making 1mM =60.57/1000

to amke 20 mM 60.57/1000x20= 1.21g(500 ml)

Dissolve the tris and NaCl in about 200 ml of water and then adjust the pH with HCL and then after adjusting the pH with HCl to 7.5, make up the volume to 500 ml with distilled water.

4) for 1 litre 0f 10X TBS

NaCl =87.7 g

1M Tris-Hcl (pH 7.5)=100 ml

Nacl for 200 ml TBS

for 1000 ml its 87.7 g

for 200 ml it will be 87.7/1000x200 =17.54 g

Tris-Hcl (7.5) added is 100 ml for 1 litre TBS

Threrefore for 200 ml

its 100/1000x200=20 ml

How to prepare Tris-HCl

Disssolve 121.14 g Tris in 500 ml water and adjust the pH to 7.5 and then make up the volume to 1 litre with distilled water. This will give 1 M Tris-HCl pH 7.5 solution.

5) The protein concentration should be 2500 ng/ml to get 250 ng protein in 100 micro litre.

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