Determination of one property of GFP

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Determinationof one property of GFP

MustafaAlaesany

Groupmembers: Hannah Kissinger, Mustafa Alaesany, Ayanna Simpson

BIO346LAB REPORT

LabSection: 04

2/22/2016

Dr.Kronengold

Introduction

Theexperiment focused on the determination of one property of GFP. Itrelied on the use of three PH solutions that are then subjected to UVlight so as to ensure to establish whether they fluorescence light orthey do not. The experiment was well conducted and the specificationsincluding the materials, procedure, results and discussions are wellindicated.

Materialsand methods:

Materials

Thematerials used up in the experiment were as included Heating blockfor providing the the heat for making the temperatures of up to 700Celcius.It also included nine Eppendorftubes in which PH solutions were put. A small quantity of PH4solution was put in three Eppendorf tubes, a small quantity of PH6solution put in three Eppendorf tubes and a small quantity of PH6solution put in three Eppendorf tubes. There was also the necessityof exposing the tubes to UV light provided by the sun. A marker penwas also used to mark the tubes that contained a given solution.Pipettes were also used to stir the solutions.

Method

Preparationof the eppendorf tubes

Nineeppendorf tubes of 100 uL each were labeled based on their trialnumber and condition. The control solution to be used for theexperiment contained dilute 100 uL GFP with 100 uL TE. Threeeppendorf tubes were labeled as ‘PH 8’, three tubes were labeledas ‘PH 6’ and the remaining three tubes were labeled as ‘PH 4.’

Additionof respective PH solutions

Thesecond step involved adding 100 uL GFP of each respective pH to thetubes in relation to how they were labeled. However, the pH 8solution was not added since it is the one which was being used asthe control.

Illuminationof the tubes

Thetubes were then subjected to UV light so as to make them illuminated.The aim was to establish whether there would be some form ofillumination on the eppendorf tubes. The interpretation of theresults would depend on this aspect. This meant that if the tubescontaining GFP happened to fluorescence, then they would be accordedone point. If they failed to fluorescence, then they would be labeledas 0 points.

Placingat a controlled temperature

Thetubes were then placed in the heating block at 70 degrees celsius andheated for a total of 30 seconds. This would help to maintain thetemperature of the optimum operation of the experiment.

Repetitionof the third step

Thethird step was repeated so as to improve the quality of the resultsobtained.

Results:

Themeasurements of the 9 bands were taken and the results are as shownin the table below.

Lane 2

Bands

ID of Standards (kDa)

Distance Migrated (mm)

Dye Migration Front (mm)

Rf

(Green + Heat) Flourescent Bande Migration (mm)

Dye Front Migration (mm)

Rf

1

82

28

2

64

35

3

49

40

4

37

44

5

26

47

6

19

51

7

15

55

8

46

58

9

32

61

Withno heat

Sample type

Minute 1

Minute 2

Minute 3

Minute 4

Minute 5

pH 8

Yes

Yes

Yes

Yes

Yes

pH 6

Yes

Yes

Yes

Yes

Yes

pH 6

Yes

Yes

Yes

Yes

Yes

pH 4

Yes

Yes

Yes

Yes

Yes

pH 4

Yes

Yes

Yes

Yes

Yes

Theresults show that there is a progressive increase in the distancetravelled within the eppendorftubes. Both the eppendorf tubes tubes containing the PH6 and PH4solutions also seem to bear a positive show of fluorescence whenthere is heating as well as when there is no heating. This clearlyshows that the GFP is clearly manifested in the solutions.

Westernblot

Thewestern blot findings showed that there was fluorescence on theexponential while there was no fluorescence on the treadline

Treadline

Exponential

SDS-PAGE

Conclusion

Theapplication of GFP effect is well commonly used as a reporter ofexpression of various genes. This is because it is able tofluorescent when it is exposed to UV light thereby showing theavailability of genes in various animals. Through the properapplication of GFP, it is quite possible to identify the differencebetween various genes that are shown by animals and what brings aboutthese differences. (In Gupta et. al, 2013)

References:

Labmanual

Herold,K. E., &amp Rasooly, A. (2009).&nbspLab-on-a-chiptechnology: Volume 2.Wymondham: Caister Academic.

Literature

InGupta, V. K., In Tuohy, M. G., In Ayyachamy, M., In Turner, K. M., &ampIn O`Donovan, A. (2013).&nbspLaboratoryprotocols in fungal biology: Current methods in fungal biology.

Molecularbiology of the cell.(2014). Place of publication not identified: Garland Science.

Acknowledgement

Iam grateful to Dr. Kronengoldfor the support that he has accorded me throughout this course inensuring that I am able to grasp the much relevant information thatis necessary for developing an apt laboratory experiment. I also takethis chance to thank my group members who include Ayanna Simpson andHannah Kissinger for the coordination that they showed during thisventure.

Table1: With no heat

Fluorescence(Y/N) 1

Fluorescence(Y/N)2

GFP (pH 8) + 100 uL TE

Y

N/A

pH6

Y

Y

pH4

Y

Y

Table2: With heat

Fluorescence(Y/N) 1

Fluorescence(Y/N)2

GFP (pH 8) + 100 uL TE

Y

N/A

pH6

Y

Y

pH4

Y

Y

Figure1: DistanceMigrated vs ID of Standards (kDa)

Figure2: More fluoriscence Figure 3:

Exponentialtreadline

Y=241 n(x)- 820

R2=0.9584

Figure1:Exponential treadline

Table1: : Rf vs Molecular weight

ID of Standards (kDa)

Rf

82

0.45

64

0.92

49

0.96

37

0.95

26

0.83

19

0.89

15

0.98

46

0.53

32

0.71

Y=-97.08In(x)-7.3732

R2=0.8772

Figure2: Molecular weight vs Rf

Reflection

Ingeneral, the experiment was quite effective at ensuring that Ideveloped the necessary skills for carrying out an experiment inrelation to determination of one property of GFP. This helps toensure that I am able to establish the effect of UV lights on variousGFP solutions so as to establish whether there is a fluorescence whenthey are exposed. The information shall be quite essential atenabling me show how various genes do express themselves.