Biology 2290F/G Lecture Notes - Lecture 13: Spectrophotometry

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carmineeagle533

5 Jan 2020

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Explain how to estimate the number of cells/ml with: a haemacytometer and a spectrophotometer where the # cells/ml = a400 /slope of standard curve. Each grid is composed of 9 large squares with the most complex one in the entre. For the size of cells we will be using we will only use squares in the centre of the grid. The centre grid, which is 1 mm x 1 mm, is further subdivided into 25 smaller squares each of which is 0. 2 mm x 0. 2 mm. Area of a square = 0. 2 mm x 0. 2 mm = 0. 04 mm2. When the glass cover is in place, the depth of the chamber above the grid is 0. 1 mm. Volume above a square = 0. 04 mm2 x 0. 1 mm = 0. 004 mm3 (1 ml = 1 cm3 = 1000 mm3) 0. 004 mm3 i. e. the volume above a 0. 04 mm2 square is 1/250,000 ml.

Related Questions

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

Calculations on Multiple Stock Solutionsâbe 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 Dilutionsâbe 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 Dilutionsâbe 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 badâ¦ 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 Dilutionsâbe 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 badâ¦ 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?

fill the blanks with the terms given

Terms: 0.21, 21, 78, 760, altitude, blood, Boyle's, Dalton's,decimal, decreased, diaphragm, directly, external intercostal,Henry's, into, inverse, lowers, out of, oxygen, parasternal,partial, percent concentration, pressure, solubility, temperature,total, volume

_________ law states that there is a/an ________ relationshipbetween [listed alphabetically] the _________ of a gas and the___________ of the gas. That is, an increase in one of the factorsis associated with a decrease in the other factor and vice versa.Thoracic cavity size determines the volume of the air in the lungs.Thus, this law is fundamental to explaining why an increase in thethoracic cavity size due to expansion of the rib cage in thehorizontal [front-to-back and side-to-side] directions viacontraction of the ___________ and _________ muscles, (listedalphabetically) and vertical [up-and-down] direction viacontraction of the ___________ causes a resultant decrease inintrapulmonary pressure, creating a pressure gradient that drivesair _____________ the lungs. Conversely, it explains why when thesize of the thoracic cavity is _________ by relaxing the musclesused during inspiration, the pressure of the gas in the lungsincreases, exceeding atmospheric pressure, creating a pressuregradient that then forces air _________ the lungs ________ lawstates that the _______ pressure of a mixed gas is equal to the sumof the ___________ pressures exerted by each of the individualgasses in the mix, which are exerted independent of one another.This law is important to pulmonary physiology because theatmospheric air we breath is a mixed gas that contains roughly__________% nitrogen and _______% oxygen. Moreover, according toHenry's law, it is the partial pressure of the oxygen in the air webreath that is the main determinant of how much of the air's oxygenwe can drive into our blood and make available to the body. A gas'spartial pressure can be calculated by multiplying the totalpressure of the mixed gas by the gas's ____________ in the mixedgas. Thus, the partial pressure of oxygen, at sea level isdetermined by multiplying total atmospheric pressure at sea level -which equals ___________ millimeters of mercury (denoted mm Hg) by________ - oxygen's percent concentration in atmospheric air,expressed as a ____________. ________ law actually states that theamount of gas we can drive into solution is _________ dependentupon 3 factors. These 3 factors are 1) the solubility of theparticular liquid for the particular gas you are considering, 2)the temperature (more gas can be driven into liquids at lowertemperatures than at higher temperatures) and 3) the partialpressure of the gas under consideration. In the body, theparticular gas we are interested in driving into a fluid is__________ and the particular fluid we are interested in drivingthe gas into is our ___________. Because blood has a particularsolubility for oxygen - and this is constant, and because thehomeostatic mechanisms in the body keep body temperature relativelyconstant, this means the single most important factor thatdetermines how much oxygen can be driven in to our blood is thepartial pressure of the oxygen in the air we breath. At sea level -the partial pressure of oxygen in atmospheric air = 760 x 0.21 =159 mm Hg. However, if one climbs to the peak of Mt. Everest, wherethe air is still a mixed gas that contains only ~21% oxygen, butthe air there is 'thinner' i.e. there is just less of it up there,the total pressure of the gas drops to about 250 mm Hg, whichsignificantly __________ the the partial pressure of oxygen and theamount of air that can be forced into the blood at that altitude.Thus this gas law explains why many individuals initiallyexperience ____________ sickness when they vacation in the Rockiesor the Swiss Alps but normally live at much lower elevations.

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Biology 2290F/G Lecture Notes - Lecture 13: Spectrophotometry

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