Report on Bio-molecular Workshop by Speakers:

Polymerase Chain Reaction (PCR) & DNA Fingerprinting

 Date               : 28/8/2010

 Time               : 9.00am-5.00pm

 Venue             : INTI International University College, Nilai

 Organizer       : Prof Dr Ang of INTI International University College

 Speaker          : Dr Geetha & Dr Choo

 Participants    : SMJK Yu Hua’s 6AS3, 6BS3, 5B students lead by Madam Sang Fay Lee

 Reporters        : Tan Zhi Qian 6BS3 , Chong Jun Ming 6BS3

 Briefing by Dr. Geetha & Dr Choo

-          Introduction of Polymerase Chain Reaction (PCR) which is used to amplify DNA strains.

-          Explain the overview of the experiment.

-          Identify the objectives and the outline of learning through the experiment.

-          Signed an agreement form that indicate all students were agreed to use their own cheek cell DNA for the PCR experiment.

 Introduction to Techniques of Using Micropipettor

-          Procedures of using a pipettor are as below:

1.      Set the desired volume by turning the centrally located rings clockwise to increase volume or counterclockwise to decrease volume.

2.      Place a tip on the discharge end of the pipettor.

3.      The plunger will stop at two different positions when it is depressed. The first of these stopping points is the level of depression that will result in the desired volume of solution being transferred. The second point is used for the complete discharging of solutions from the plastic tip.

4.      Depress the plunger until you feel the initial resistance which is the first stopping point and insert the tip into the solution, just barely below the surface of the liquid and not as deep as possible.

5.      Carefully and slowly release plunger.

-          Students were giving a chance to adapt the techniques using a pipettor to draw different amount of solutions for about 15 minutes

-          Students practice by writing the volume for different types of pipettor in the manual given earlier.

Isolation of Cheek Cell DNA

 -          Students were requested to obtain cheek cell from the saliva.

-          Procedures for the isolation of cheek cell DNA:

1.      Each student was provided with a paper cup containing 10ml of 0.9% NaCl solution. Every student was requested to label the cup with their own initial/name.

2.      The solution was place in the mouth and rinse thoroughly to loosen and free cells from the epithelial lining. Vigorous “sloshing” was carried out for about 1 minute.

3.      The solution was transfered from the paper cup to the 15ml centrifuge tube.

4.      The tube was placed in the benchtop centrifuge located in the laboratory. All the participants’ samples had been collected and being centrifuge at 6000rpm for 10 minutes.

5.      The supernatant was removed without disrupt the pellet and cells at the bottom of the tube.

6.      0.75ml of Chelex@100 resin was added to the tube containing the pellet and cells using a pipettor.

7.      The pellet was thoroughly broke up in the Chelex suspension using a pipettor.The suspension was then transferred back to the 1.5ml tube. Number/Initial/Name was placed on the top of the lid using a fine-point marker pen.

8.       The tube is then placed in one of the holes of a dry bath that has been adjusted to 95°C. The tube was incubated for 10 minutes. The heat treatment serves to lyse the cells, denature proteins, inactivate nucleases, and release the cells’ DNA into solution.

9.      After the treatment, the tube was transferred to ice for one minute, and then placed in a microcentrifuge. All the samples were centrifuged for 45 seconds.

10.  The tube was placed on ice.

Preparation Of PCR Cocktail & Thermal Cycling

-          A convenient way to set up PCR is to use PCR beads, which have all the enzymes, buffers, and dNTPs necessary for the reaction.

1.      5 µl of student DNA, 10µl of forward primer and 10 µl of reverse primer were added into a tube. The solution was gently mixed by tapping lightly the tube on the table. The tube was then placed back on ice.

2.      (a)  The thermocycler (Bio-Rad Mycycler™ thermal cycler) was powered up by pressing the Standby key on the front panel of thr instrument.

(b) F1- Protocol Library was pressed to access the Protocol Library. Protocol Genetics 1 was selected by using the arrow keys and pressing the Enter key.

·         The Genetics 1 protocol has been adjusted to the following cycling schedule:

i.                    3 minutes “hot start” at 94°C

ii.                  30 cycles of the following:

§  1 minute 94°C

§  1 minute 65°C

§  1 minute 72°C

Finish at 72°C for 10 minutes

             (c) Selection menu appeared. “Run Protocol” was selected.

             (d) F5-Begin Run was pressed to begin the run.

  3.   We learnt a PCR Song Sing Along session while the thermo cycling was on- going.

Having Lunch At INTI University College Cafeteria

-          We had our lunch sponsored by INTI International University College Cafeteria at about 1.00pm.

Agarose Gel Electrophoresis

A. Preparing the Gel

1. 0.75 g of agarose was weighed into a 250ml culture bottle.
2. 50ml of electrophoresis buffer (TAE buffer) into the culture bottle containing the agarose. The bottle was cap loosely to release air during boiling. The agarose solution was microwave at relatively high hear for about 2 minute until the powder is completely dissolved and the agarose solution looks clear. The liquid gel was allowed to cool for about 5 minutes.
3. The electrophoresis chamber was prepared.
4. Rubber casting dams were placed on the ends of the gel tray. The tray was then placed in the chamber.
5. 8/12 comb which one side has 8 spacers and the other side 12 spacers was used to made 8 wells on the gel.
6. When the agarose solution was cool but has not solidified yet, 5µl of SYBR® Safe DNA gel stain (10,000x concentrate in DMSO) to the 50ml of agarose solution. The bottle was swirled gently. The molten agarose solution was then carefully poured onto the center of gel tray.
7. The gel was protected from light by covering it with box.
8. The comb and rubber end blocks were removed once the gel has solidified.
9. The chamber was slowly filled with electrophoresis buffer (350ml) until the upper surface of the gel is submerged about 1mm.

B. Loading the Samples

-          The gel was loaded starting from left hand side.

-          4 µl of 6x loading dye was added into the PCR tube. The lid of the PCR tube was secured and gently mixed by tapping on the table.

-          Pipettor was adjusted to 20 µl and attached to a clean tip. 20µl of DNA sample were drawn into the pipette tip.

-          After loading the sample into the pipette tip, placed it into the buffer and above the gel well.

-          Do not put the tip too deeply into the gel well because it can puncture through the well and the sample will leak out.

-          The sample is ejected smoothly into the well.

-          Dr Geetha placed 10µl of commercial low-molecular-weight ladder into the right outermost well.

-          After all the samples had been loaded, the power supply was set at 200V and turned on for 5 minutes to “stack” the DNA against the wall of the gel well, then the voltage was reduced to 75V for the remaining gel run.

C. Viewing the Gel

-          A small notch was made at one side of the gel bottom with a spatula to avoid confusion as to which is the first and last lane.

-           The gel tray were lifted from the electrophoresis chamber and the gel was carefully slide from the gel tray into the UV light chamber. All these were carried out by wearing gloves.

-          We observed our gel under UV light. Photograph of the gel was taken.

We were very lucky to attend the workshop for free.. Even though most of us didn’t really understand the experiment fully but we have learnt a lot on biotech, we were exposed to the techniques to obtain DNA fingerprinting. This workshop really sparks a great interest in many of us to venture into biotech. We promised to study hard to be one of the biotech students. Many many thanks to Dr Ang who is generous in providing such facilities and opportunities for us. We left INTI International University College and reached school at about 5.00pm.

6BS3’students having a photo after reaching INTI International University College,Nilai

Teacher & Students heading to the lab

6BS3’ students putting their belongings in the shelf provided.

Students were given a chance to know how to use a micropipettor

Dr Geetha introducing the centrifuge machine

Saliva after centrifuged.

Incubator

Thermal Cycling

Loading the gel

Viewing the gel

Mdm Sang presenting the souvenir

Mdm Sang presenting souvenir

Mdm Sang was giving souvenir

Group Photo

Prepared by :
Tan Zhi Qian (6BS3)
Chong Jun Ming (6BS3)