Table 2: shows the collected samples and the source

Type of samples Number of samples Source of sample Bladder cancer

samples

Urine Samples Twelve Theodor Bilharz YES

Research Institute

Urine Samples Three Theodor Bilharz NO

Research Institute

2.1.1 Chemicals requirements

Table 3: shows the used chemicals, number of kits and the source.

Chemical Kit number Source of kit

Elution buffer #T1016L Promega Inc

 Lysis solution #MD130A Promega Inc

Magnetic beads #MD205A Promega Inc

Distilled water / Theodeor Bilharz Institute.

Ethiduim / Sigma USA

Agarose powder / Sigma USA

Standard DNA ladder / Promega Inc

DNA loading dye / Promega Inc

 EDTA / Promega Inc

10X PCR buffer #N8080006 Promega Inc

10mM dNTPs #N0446S Promega Inc

25mM MgCl2 #R0971 Promega Inc

Taq DNA polymerase enzyme / Promega Inc

TaqMan® Universal Master Mix #4440043 Promega Inc

II 2X

TaqMan® Assay 20X #4441114 Promega Inc

DNase –free water #AM9922 Promega Inc

2.1.1 Equipment
Table 4: Shows the used equipment and the source.

Equipment Source

Vortex Cole-Parmer

Hot block The lab depot, Inc

Incubator Shellab

Centrifuge Eppendorf

Microcentifiuge Star Lab

Laminar flow JSCB-1200SB Thermo scientific USA.

NanoDrop 2000C spectrophotometer Thermo scientific USA.

Electrophoresis TT-HES-1 Hercuvan, India.

UV-Trans illuminator Scintific Ltd, UK.

 Microwave 750W Moulinex, Egypt

7500–PCR AB Applied Biosystems, Foster city, CA

2.1.1 Disposal Materials

Table 5: Shows the used Disposal Martials used

Equipment Source

Microcenterifuge tube Star lab

Tips Star lab

Pipettes Star lab

 Non-powder gloves Star lab

Tube racks Star lab

Dropper Star lab

Non- additive tube Star lab

Table 6: Reaction setup of PCR reaction per one sample.

Component Volume

Taq DNA polymerase 2 μl

10X Buffer 10 μl

25mM Mgcl2 10 μl
 20mM dNTPs mix 1 μl

TP53 exon 4 forward primer 2 μl

TP53 exon 4 reverse primer 2 μl

DNA template sample 10 μl

dH2O Up to 50

total 50 μl

Table 7: shows the Cycling conditions of PCR

:

Cycle Step Temp Time Cycles

Initial 95°C 30 seconds 1

Denaturation

Denaturation 95°C 30-60 seconds 30

Annealing 58°C 30-60 seconds 30

Extension 68°C 1 minute per kb= 4 min 30

Final Extension 72°C 5 minutes 1

Hold 4°C ∞

2.1.1 Gel Electrophoresis analysis

Table 8: shows the preparation of 50X TAE Electrophoresis buffer.

Reagent Volume

Tris Free base 242g

Disodium EDTA 18.61 g

Glacial Acetic acid 57.1 g

Deionized / Milli-Q Water Up to 1 Litter

Table 9: shows the mean value of age, creatinine, and urea for the all samples.

Value of Mean

Age 67

Creatinine 1.2

Urea 37
Table 10: show the patient data and volunteer's data.

Patient Gender Age Type of Bladder Diabetic Smoking Bilharzia Creatinine Urea level
number sample cancer patient person Infection level
patient
1 Male 50 Urine NO NO NO NO 1 ml/dl 9 ml/dl
Sample
2 Male 55 Urine NO NO NO NO 0.9 ml/dl 10 ml/dl
Sample
3 Male 60 Urine NO NO NO NO 1.1 ml/dl 7 ml/dl
Sample
4 Male 82 Urine YES NO NO NO 1.2 ml/dl 39 ml/dl
sample
5 Male 53 Urine YES NO YES YES 1.3 ml/dl 74 ml/dl
Sample
6 Male 73 Urine YES YES NO NO 1.1 ml/dl 32 ml/dl
Sample
7 Male 71 Urine YES NO YES NO 1.4 ml/dl 49 ml/dl
Sample
8 Female 71 Urine YES NO NO NO 0.7 ml/dl 37 ml/dl
Sample
9 Female 86 Urine YES NO NO NO 1.89 ml/dl 58 ml/dl
Sample
10 Male 66 Urine YES NO NO NO 1.5 ml/dl 40 ml/dl
Sample
11 Male 70 Urine YES NO YES NO 1.5 ml/dl 23 ml/dl
Sample
12 Male 85 Urine YES NO NO YES 1.2 ml/dl 37 ml/dl
Sample
13 Male 69 Urine YES YES NO NO 0.8 ml/dl 29 ml/dl
Sample
14 Male 67 Urine YES NO YES NO 1.5 ml/dl 28 ml/dl
Sample
15 Male 60 Urine YES NO YES NO 1.8 ml/dl 84 ml/dl
Sample
Table11: shows the clinical data for control healthy volunteers (3

volunteers).

Mean age 55

Mean creatinine level 1

Mean urea level 8.6

Table 12: shows the clinical data for bladder cancer patients with their

mean values (12 patients).

Mean of age 71

Mean of creatinine level 1.32

Mean of urea level 44

Smokers (number) 5

Bilharzia infection (number) 2

Sample Concentration Purity Source

number

1 12.1 ng/µl 2 Control

2 18.5 ng/µl 1.7 Control

3 26.3 ng/µl 2 Control

4 10 ng/µl 3.8 Bladder cancer patient

5 9 ng/µl 3 Bladder cancer patient

6 8.3 ng/µl 5.4 Bladder cancer patient

7 Negative Negative Bladder cancer patient

8 132 ng/µl 2 Bladder cancer patient

9 7 ng/µl 4 Bladder cancer patient

10 532ng/µl 1.6 Bladder cancer patient

11 Negative Negative Bladder cancer patient

12 89 ng/µl 2.3 Bladder cancer patient

13 158.6 ng/µl 2.6 Bladder cancer patient

14 Negative Negative Bladder cancer patient

15 145 ng/µl 2.3 Bladder cancer patient

Exons Primers Annealing

Tempreture (OC)

4 5': 58 OC

Forword CTGGTCCTCTGACTGCTC

TT 3'

4 5': 58 OC

Reverse AGGCATTGAAGTCTCATG

GA 3'
 Figure 10: shows the lanes that present the amplification of Tp53 exon 4 in patients.

Figure 11: shows the lanes that present the amplification of Tp53 exon 4 in patients.

*ST: standard= marker, C: control sample, Pm: patient with mutation, P: patient.