BS2011 Practical & Employability Skills Density Gradient Centrifugation

26 Jun BS2011 Practical & Employability Skills Density Gradient Centrifugation

uestion
Aim Centrifugation (MCQ)

(Points: 1)

Which of the following choices best summarises the scientific aim of the experiment?

a. To determine the density of the blue beads by using isopycnic centrifugation to separate beads of differing densities and observing their relative Rf values

b. To separate beads of differing densities by isopycnic centrifugation

c. To demonstrate the method of differential centrifugation

d. To utilise beads of differing densities to simulate a complex biological mixture and to demonstrate the recording of Rf values in differential centrifugation

e. To demonstrate the method of isopycnic centrifugation

f. To separate beads of differing densities by differential centrifugation

g. To demonstrate that isopypnic centrifugation can separate particles of differing densities and so is useful as an analytical technique

h. To utilise beads of differing densities to simulate a complex biological mixture and to demonstrate the recording of Rf values in isopycnic centrifugation

i. To determine the density of the blue beads by using differential centrifugation to separate beads of differing densities and observing their relative Rf values

j. To use isopycnic centrifugation to perform a differential separation of beads of different densities

k. To demonstrate that differential centrifugation can separate particles of differing densities and so is

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

2.Rotor radius

(Points: 1)

From the information given in the practical schedule it is possible to deduce that the radius of the RPT70T rotor is approximately

a. 6.8 x 10^-5 cm

b. 0.007 cm

c. 10 cm

d. 116 cm

e. 7 cm

f. There is not enough information to deduce this.

g. 113840 cm

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

3.RCF (T/F)

(Points: 1)

The relative centrifugal field is expressed in the unit g, where g represents mass in grams

True
False

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

4.G force

(Points: 1)

When the centrifuge was run at 16500 rpm an RCF of 21000g was exerted, therefore when the centrifuge was run at 2000 rpm after the beads were added approximately what RCF was exerted?

a. The answer requires knowledge of the radius which cannot be determined from data given in the schedule

b. 2545g

c. 21000g

d. 310g

e. 14500 rpm

f. 19000g

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

5.Title of the graph1

(Points: 1)

A suitable title for the graph would be

a. Differential centrifugation

b. mean distance travelled (cm) vs bead density (g/cm3)

c. Rf values

d. bead density (g/cm3)

e. Rf values vs bead density (g/cm3)

f. mean distance travelled (cm) vs total distance (cm)

g. Experimental results

h. distance travelled by beads (cm)

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

6.Smallest standard deviation

(Points: 1)

The smallest standard deviation is seen in the data for which beads?

a. Pink

b. Violet

c. Green

d. Yellow

e. Orange

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

7.Standard deviation of violet beads

(Points: 2)

What is the standard deviation of the distance moved by the violet beads? Choose the range that fits most closely with your answer.

a. 0.29 – 0.57

b. 0.5 – 0.7

c. 0.29 – 0.38

d. 0.36 – 0.39

e. 0.50 – 0.57

f. 0.7 – 0.9

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

8.Average Rf of the pink beads (2012)

(Points: 2)

The average Rf for the pink beads lies between

a. 0.75 and 0.77

b. 0.80 and 0.85

c. 1.25 -1.28

d. 0.75 and 0.80

e. 0.81 and 0.83

f. 1.20 and 1.28

g. 0.78 and 0.80

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

9.Origin

(Points: 1)

There is an error in the experimental data because the line of best fit does not go through the origin (0,0).

True
False

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

10.Outliers

(Points: 1)

An outlier is a data point that appears inconsistent with the overall data and with caution may be removed from the analysis. Write the colour of any beads that may be considered outliers.

1.

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

11.Density of the unknown blue beads

(Points: 3)

Choose the density range which is the best fit with the density of the unknown beads:

a. 1.15 and 1.20

b. 1.74 and 1.85

c. 1.085 and 1.10

d. 1.065 and 1.095

e. 1.040 and 1.065

f. 1.00 and 1.20

g. 1.05 and 1.15

h. 1.70 and 1.95

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

12.Centrifugation – Discussion

(Points: 3)

Discuss the data obtained, considering the statistics you performed, the graph drawn and the Rf value of the unknown.

Paragraph

Insert equation.gif” alt=”Go”>

Save Answer.gif” alt=”http://uelplus.uel.ac.uk/webct/images/shim.gif”>

13.Centrifugation Conclusion

(Points: 2)

Write a one sentence conclusion of the experimental findings

Paragraph

bead

Data obtained by each group G1-G6 (distance migrated in cm)

Band

G1

G2

G3

G4

G5

G6

mean

s.d.

Rf

density

Yellow

1.3

1.2

1.1

1.3

1.3

1.6

1.35

0.18

0.23

1.033

Green

0.8

0.6

0.5

0.8

1

1.1

0.80

0.23

0.12

1.051

Red

2.9

2.8

2.4

2.4

2.5

3.1

2.68

0.29

0.41

1.063

Blue

3.5

3.4

3.1

3

2.9

3.7

3.27

0.31

0.50

Orange

4.3

4.3

3.7

3.6

3.6

4.4

3.98

0.39

0.60

1.086

Brown

4.8

4.6

4.3

4.1

4

5

4.47

0.40

0.68

1.098

Pink

5.5

5.5

4.9

4.8

4.7

5.7

5.18

0.43

0.78

1.119

Violet

5.9

6

5.5

5.6

5.5

6.3

5.80

0.32

0.88

1.138

Notes.

The table contains the results for the distances migrated by the various coloured bands of beads, obtained by six different groups G1-6. The total distance to the bottom of the tube is 6.6 cm (needed for the Rf calculation). The Blue band is the unknown, for which you have to find its density. s.d. = standard deviation.

.05in;mso-layout-grid-align:auto”>BS2011 Practical & Employability Skills
Density Gradient Centrifugation

Introduction

When a suspension of particles is centrifuged, the sedimentation rate of a particle is proportional to the forceapplied. The physical properties of the solution/medium will also affect the sedimentation rate. At a fixedcentrifugal force and liquid viscosity, the sedimentation rate is proportional to the size of the particle and thedifference between its density and the density of the surrounding medium.

In this practical, isopycnic density gradient centrifugation has been used, in which the medium employs a gradient to assist the separation. In isopycnic centrifugation, a density gradient which includes all the densities of the sample particles is used. Eachparticle will sediment through the gradient until it reaches an equilibrium position where the density of the particleis equal to the density of the surrounding medium. In this experiment, a mixture of coloured beads (which have different densities by colour) are placed in this density gradient and centrifuged. These beads separate into a series of coloured layers in the centrifuge tube, each layer corresponds to the density of that particular colour of bead, irrespective of size.

Method:

To pre-form the density gradient, a volume of 9.5 ml of a 60% solution of percoll was added to two polypropylene centrifuge tubes and balancedaccurately. (Remember when balancing these tubes the weight of the adapters and tube caps must betaken into account). The tubes were capped and placed in the rotor of the centrifuge indiametrically opposing holes. The rotor was then spun for 45 minutes at a speed of 16,500 rpm. (For this rotor, RPT70T, this speedcorresponds to a g-force (RCF) of approximately 21,000g).

To run the experiment:

· A volume of 100µl of density marker beads was gently layeredon the top of the density gradient.

· The tube containing the density layer was placed in a bench centrifuge and spun at 2,000 rpm for 10 minutes. The tube was carefully removedand the distance each band of beads had moved from the meniscus was measured. The density of one of the layers or beads is unknown, and the aim of the experiment is to find the density of the ‘Unknown’ layer.

What you are required to do

Please note carefully below what needs to go into your lab notebook or into UELPlus [as a checklist, the steps required have been underlined below]

1. In your lab notebook, do a short write-up of the experiment as if you had carried it out in the lab. You also need to do various calculations as listed below: write these calculations into your notebook, which should also contain either a drawn graph, or else a printout of a graph done in Excel (see below). You also need to input some results from the experiment into UEL Plus (see below).

2. There is a Word file (centrifugation practical data.doc) on UEL Plus. This contains the results from the experiment, giving the distance in cm that each band of beads has moved from the meniscus.

3. Calculate the mean and standard deviation for the distance moved (in cm) for each of the different coloured beads in turn and also for the ‘Unknown’. This should give you 8 sets of means and standard deviations.

4. You are also given the total distance in cm in the data file; calculate the Rf ratio for each band of beads using the mean distance travelled for each band of beads. Calculate the Rf ratio for each coloured band using the following formula: (mean distancemigrated by the coloured band/total distance).

5. Plot a graph of the Rf ratio (mean distancemigrated by the coloured band/total distance) for each coloured band against the density of that coloured band. You can either draw the graph directly into your lab notebook and draw the line of best fit through the points, or if you prefer to use Excel to plot the graph, you must then print out a copy of the Excel graph and include the line of best fit of the points and fix it into your lab notebook.

6. Use the graph to determine and record in your notebook the density of the unknown (the orange band) from the graph. Make sure you show on your graph how you have estimated the density of the ‘Unknown’.

7. ON UEL PLUS follow the instructions for the data which you should enter

DENSITIES OF COLOURED BANDS

These are the densities of the various coloured bands in gm/cm3, which you will need in making the graph. The density should be plotted on the graph as the horizontal (x) axis.

BAND DENSITY

Yellow 1.033

Green 1.051

Red 1.063

Orange 1.086

Brown 1.098

Pink 1.119

Violet 1.138

Blue Unknown

Resources:

The series of books entitled ‘Practical Skills in …’ (‘…Biomolecular Sciences, ..Forensic Science’ etc) contain short sections on drawing graphs (including calibration graphs) – such graphs were included in the first year module BS1000.