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BCH 499 Research and seminar -2

 

499 كيح مشروع بحث -2

عدد الساعات : 2(0+2)

المتطلب : 232 كيح ، 498 كيح

 

وصف المقرر

وفيه يدرس الطالب بعض التجارب العملية المتقدمة مثل فصل بعض الجزيئات الحيوية ، ودراسة تأثير بعض الهرمونات على مستوى السكريات والدهون في الدم

BCH 499 Research and seminar -2

Symbol & No:

       BCH 499

 

Course Title

Research and Seminar

 

Credit Hours:

2

 

 

 

Introduction to the Course

Each student has to carry out the research project on the biochemical topic as assigned by their supervisor. The student follows the research protocol and should be able to design laboratory experiments independently or with minimal supervision. The students are required to, carry out literature search on the research topic, prepare a report and discuss their results. The results are compiled in the form of a report and also presented in a poster for evaluation by a committee. Research report should be completed by using the heading: Introduction, Methods, Results, Discussion, Conclusion and References. Research report will be submitted by the students about her relevant research topic, which will be examined by two of the faculty member.

 

Aim of The course

The aim of this course is to develop the research skills and how to present their scientific work in the form of a research report as well as poster. 

 

Objective of the course

 

 

 

 

 

 

 

 

 



1-   To learn a new laboratory method

 

2-   To analyze useful results from your data

 

3-   To be able to design laboratory experiments independently

 

4-   To be able carry out literature search on the research topic

 

Attendance

All laboratory work is to be done during the semester on Tuesday & Wednesday, 8:00 am to 4 p.m.)

Student evaluation:

 

Report + poster

Oral Presentation

Introduction lectures test

Practical work

Cleanliness of lab

Supervisor

30

30

10

 

 

Lecturer

 

 

 

20

 

Technician

 

 

 

 

10

 

Lecture Schedule for the Second Semester, Academic Year 1430-31:

Topic

Dr.

Date

Introductory lecture on Course BCH499

 How to make poster?

Dr. Samina H. Haq   

  Monday 8th-3-2010       22- 3-1431  from 12-1p.m

Quality  Control in research

Mrs Farida Alkhuwather

Monday   15-3-2010      29-3-1431   from  12-1p.m    

How to write references? 

Dr. Manal Shalabi

Monday 22-3-2010     6-4-1431     from 12-1p.m

 

  How to write practical report?

Prof. Afaf-

Mon   29-3-2010         13-4-1431   from 12-1 p.m

How to use SPSS in research?

 By Prof. Arjumand Warsy

 Mon 5-4- 2010        20-4-1431          from 12-1p.m

 

Deadline date for your experimental work & report submission  

Weak

date

Expectations from the student

Weak 3     

23 &24 /03H

Starting your experiment work by preparation of buffer

Weak 4     

30/03 & 1 /04 H

start DNA Extraction

Weak  5

 7 & 8 /04

 DNA characterization

Weak  6

14 & 15 /04 H

Submit your report

 

 

Lab Safety          Microsoft Word - Lab Safety.pdf

     

  • *      Do not eat food, drink beverages, or chew gum in the laboratory.
  • *      Do not use laboratory glassware as containers for food or beverages
  • *      A lab coat should be worn during laboratory experiments.
  • *      Be prepared for your work in the laboratory, read all procedures thoroughly before entering the laboratory. 
  • *      Never fool around in the laboratory.
  • *     Mouth pipetting is not permitted.
  • *      Do not sit on the lab benches
  • *      Handle and dispose of hazardous chemicals properly
  • *     Be aware of objects that can burn or give electrical shocks.
  • *      Be familiar with location and use of safety items.
  • *     Wipe your lab bench with a damp sponge at the end of each lab period
  • *     Never work alone
  • *     Sweep up broken glassware as soon as possible.
  • *     All solutions that you prepare must be labeled with date, your name, and the type and concentration of the reagent

*  When chemicals are spilled they should be wiped or swept up or both as soon as possible

 Experimental Project:

 

 

Extraction of DNA from Whole Blood

John M. S. Bartlett and Anne White

1. Introduction

There are many differing protocols and a large number of commercially available kits used for the extraction of DNA from whole blood. This procedure is one is used routinely in both research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tissues or cell cultures (omitting the red blood lysis step.

2. Materials

This method uses standard chemicals that can be obtained from any major supplier;

we use Sigma.

1. Waterbath set at 65°C.

2. Centrifuge tubes (15 mL; Falcon).

3. Microfuge (1.5 mL) tubes.

4. Tube roller/rotator.

5. Glass Pasteur pipets, heated to seal the end and curled to form a “loop” or “hook” for spooling DNA.

6. EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilled water. Adjust pH to 8.0 with NaOH pellets (this will require about 20 g). Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min.

7. 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water. Adjust pH with concentrated HCl (this requires about 60 mL).

8. Reagent A: Red blood cell lysis: 0.01M Tris-HCl pH 7.4, 320 mM sucrose, 5 mM MgCl2, 1% Triton X 100.

9. Add 10 mL of 1 M Tris, 109.54 g of sucrose, 0.47 g of MgCl2, and 10 mL of Triton X-100 to 800 mL of distilled water. Adjust pH to 8.0, and make up to 1 L with distilled water. Autoclave at 10 p.s.i. for 10 min (see Note 1).

10. Reagent B: Cell lysis: 0.4 M Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodium dodecyl sulphate, pH 8.0. Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA (pH 8.0),

8.76 g of NaCl, and adjust pH to 8.0. Make up to 1 L with distilled water. Autoclave 15 min at 15. p.s.i. After autoclaving, add 10 g of sodium dodecyl sulphate.

11. 5 M sodium perchlorate: Dissolve 70 g of sodium perchlorate in 80 mL of distilled water. Make up to 100 mL.

12. TE Buffer, pH 7.6: Take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, and make up to 1 L with distilled water. Adjust pH to 7.6 and autoclave 15 min at 15. p.s.i.

13. Chloroform prechilled to 4°C.

14. Ethanol (100%) prechilled to 4°C.

 

 

 

3. Method

3.1. Blood Collection

1. Collect blood in either a heparin- or EDTA-containing Vacutainer by venipuncture (see Note 2). Store at room temperature and extract within the same working day.

3.2. DNA Extraction

To extract DNA from cell cultures or disaggregated tissues, omit steps 1 through 3.

1. Place 3 mL of whole blood in a 15-mL falcon tube.

2. Add 12 mL of reagent A.

3. Mix on a rolling or rotating blood mixer for 4 min at room temperature.

4. Centrifuge at 3000g for 5 min at room temperature.

5. Discard supernatant without disturbing cell pellet. Remove remaining moisture by inverting the tube and blotting onto tissue paper.

6. Add 1 mL of reagent B and vortex briefly to resuspend the cell pellet.

7. Add 250 μL of 5 M sodium perchlorate and mix by inverting tube several times.

8. Place tube in waterbath for 15 to 20 min at 65°C.

9. Allow to cool to room temperature.

10. Add 2 mL of ice-cold chloroform.

11. Mix on a rolling or rotating mixer for 30 to 60 min (see Note 3).

12. Centrifuge at 2400g for 2 min.

13. Transfer upper phase into a clean falcon tube using a sterile pipet.

14. Add 2 to 3 mL of ice-cold ethanol and invert gently to allow DNA to precipitate (see Note 4).

15. Using a freshly prepared flamed Pasteur pipet spool the DNA onto the hooked end (see Note 5).

16. Transfer to a 1.5-mL Eppendorf tube and allow to air dry (see Note 6).

17. Resuspend in 200 μL of TE buffer (see Notes 7 and 8).

4. Notes

1. Autoclaving sugars at high temperature can cause caramelization (browning), which degrades the sugars.

2. As will all body fluids, blood represents a potential biohazard. Care should be taken in all steps requiring handling of blood. If the subject is from a known high risk category (e.g., intravenous drug abusers) additional precautions may be required.

3. Rotation for less than 30 or over 60 min can reduce the DNA yield.

4. DNA should appear as a mucus-like strand in the solution phase.

5. Rotating the hooked end by rolling between thumb and forefinger usually works well. If the DNA adheres to the hook, break it off into the Eppendorf and resuspend the DNA before transferring to a fresh tube.

6. Ethanol will interfere with both measurements of DNA concentration and PCR reactions. However, overdrying the pellet will prolong the resuspension time. The small amount of EDTA in TE will not affect PCR. We routinely use 1 μL per PCR reaction without adverse affects.

8. DNA can be quantified and diluted to a working concentration at this point or simply use 1 μL per PCR reaction; routinely, it is expected                                                                                              200 to 500 ng/μL DNA to be the yield of this procedure

  

DNA Characterization

Measure DNA purity

One of the most common methods for nucleic acid detection is the measurement of solution absorbance at 260 nm (A260) due to the fact that nucleic acids have an absorption maximum at this UV wavelength. compounds commonly used in the preparation of nucleic acids absorb at 260 nm leading to abnormally high quantitation levels. However, these interference and preparation compounds also absorb at 280 nm leading to the calculation of DNA purity by performing ratio absorbance measurements at A260/ A280.

The ratio can be calculated after correcting for turbidity (absorbance at 320nm).

DNA Purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)

A260/A280 = 1.7 to 2.0 for “pure” DNA

Therefore, constructing an absorbance ratio between these two absorbance wavelengths can provide an estimate of sample purity. As a general rule any preparations with an A260/A280 greater than approximately 1.7 is called “pure”

Material:

1.      DNA sample

2.      SSC buffer:

10X: 0.5 M sodium citrate ( 14.71 gm of Na3 citrate , dissolve and make it up to 100 ml in H2O)

3.      Spectrophotometer

4.      Quartz cuvettes

Method:

1. Dissolve your DNA in 3 ml of 0.1X SSC.

2. Read the absorbance of DNA at different wave lengths starting at 220 nm up to 300 nm using 0.1 X SSC as your blank in each of the readings

3. Plot the absorbance spectrum of your sample against each wave length

4. Compute the absorbance ratio 260:280

                    

Measure DNA Concentration:

  

DNA concentration can be estimated by measuring the absorbance at 260nm (A260), adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor

Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml

concentration = 260 reading * Absorbance factor * dilution factor
Absorbance Factor
ssDNA: 37 µg/ml
dsDNA: 50 µg/ml
ssRNA: 40 µg/ml

Prepare  A blank cuvette by pipetting 2000μl of 0.1X SSC  Into a cuvette.

Then add Five or ten μl of samples into other cuvettes, then add 1990 or 1995μl of 0.1X SSC

The dilution factor = (sample volume + 0.1X SSC volume) / sample volume = either 200 or 400.

Measure the Concentration of DNA using spectrophotometer at 260 nm and calculate the 260nm/280nm ratio

C = ε × A × L × dilution factor                            

Where: C = Concentration ng/μl or μg/ml

ε = Extinction coefficent

A = Absorbance at 260 nm

L= Length of quvette

 

Total yield is obtained by multiplying the DNA concentration by the final total purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (ml)

 

MELTING DNA

 

"Melting", refers to the breaking of the hydrogen bonds that hold both of the DNA  helices  together.  This  is  a  curve  of  the  absorbance  values  at  260  nm  for  increasing temperatures. As might  be  expected,  increasing  the  temperature  tends  to  cause  chemical  bonds  to  break. This  would  cause  DNA  to  be  converted  from  a  double  stranded  to  a  single  stranded configuration.  The absorbance value of single stranded DNA is greater than the double stranded form.

In this laboratory excursion, you will determine if a sample contains DNA by gathering data to plot what  is  known  as  a melting  curve.    The  data  results  from  a characteristic  change  in  the absorbance  values  at  260  nm  that  are  caused  by  an  increase  in  temperature. You  can  use  the spectrophotometer and the circulating water bath to determine the melt curve of a sample of DNA.

Double stranded DNA has a  lower absorbance value  than  the single stranded form at 260 nm. In the space below, draw a predicted absorbance value curve for a sample of DNA as it is slowly heated.

 

 

Method:

1. Dissolve your DNA in 3 ml of 0.1X SSC

2. Read the absorbance at 260 nm making sure that its between 0.8-1.0, if it was higher then dilute till you reach the desired absorbance.

3. Put the diluted DNA sample in a water bath of 30°C for about 5 minutes to equilibrate then read its absorbance at 260 nm using 0.1 X SSC as your blank

4. Repeat step 3 at each of the following temperatures:

30. 40. 50. 60. 70, 80 and 100° C

5. Plot a graph of your absorbance at different temperatures and determine the melting point Tm

6. Calculate the GC content of your sample

Percent of G + C = (Tm -69.3) x 2.44

Temperature degree centigrade 

Absorbance reading    at 260 nm

30

 

40

 

50

 

60

 

70

 

80

 

85

 

90

 

95

 

100

 

 

Di phenyl amine reaction:

A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95 °C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 660 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

 

Material:

 Di phenyl amine reagent:

Dissolve 1.5 gm DPA in 100 ml glacial acetic acid, add 1.5 ml sulphuric acid.

Store in a dark glass bottle.

Add 0.5 ml of acetaldehyde solution ( 1ml/50 ml water)

 

Method:

1. Prepare 9 test tubes, 1-6 (standard DNA solution) 7,8 ( test sample), and 9 blank ( sodium citrate)

2. Prepare a standard solution of DNA with a concentration of 3 mg/ml and prepare a series of dilutions in tubes 1-6

3. Add to each tube 3 ml of DPA solution

4. Place tubes in a boiling water bath for 10 min

5. Read the absorbance’s at 660 nm

6. Determine the concentration of your sample

           DNA Pictures.doc 

 

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