Jueun (Christine) Kim

MCB 251 Section V

PCR Lab Report
09/16/2016

Polymerase Chain Reaction Analysis

Procedure:

1. Wear gloves in order to protect the DNA and enzymes from degradation by enzymes on

skin and to avoid contact with Ethidium Bromide gels in Gel Electrophoresis of PCR

products.

2. Obtain the microfuge tubes placed in Ice Buckets, in which one is with 12.5 µl 2X

Paq5000 Master mix (MM), one is with 2.5 µl Template DNA (T), one is with 5 µl

Upstream Primer (UP), and the last one is with 5 µl Downstream Primer (DP).

3. Add all the given reagents into the Template DNA (T) tube, and gently pipet as adding to

the mixture.

4. Centrifuge the tube to make sure that the reagents are thoroughly mixed and concentrated

at the bottom of the microfuge tube. (Do not forget to balance the centrifuge!)

5. Dispense 25 µl from the mixture into a labeled PCR tube.

6. Place the tube in the PCR thermocycler and start the PCR cycle, which will run for about

2.5 hours.

7. After the PCR cycle is complete, run out the product on an agarose gel.

8. Obtain a precast agarose gel and place it in a gel casting tray.

9. Place the gel and casting tray in an empty gel box, and add enough 1X TBE Buffer so

that the gel is submerged completely.

10. Using the P20 micropipette, load 10 µl of DNA Standard (St) to the first well in the

agarose gel.
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11. Mix 5 µl of loading dye with 10 µl of the PCR mixture, then load all 15 µl of the mixture

into the gel well.

12. Run the agarose gel a 100 V for about 45 minutes.

13. When the gel run is done, turn off the power supply, take out the casting tray from the gel

box, and obtain a photograph of the gel.

Data/Results:

The far left lane indicates the marker DNA, 1Kb DNA Ladder, the DNA Standard (St)

loaded to the first well in the gel. It helps to guide the various DNA’s sizes and quantities by

putting a “ladder.” The second lane is not my group’s data. The third lane represents my group’s

data. There is a clearly indicated band of the PCR mixture. The size and quantity of the DNA can

be figured out by observing the brightness and the similarity of the row to the DNA Ladder.
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Discussion:

In the gel photo, there is a clear single band shown in the lane of my PCR mixture, along

with the several bands with different brightness in the first lane, the DNA Ladder’s. There is

nothing that is seen that was unexpected. I expected to see the single band that represents the

characteristics of the DNA.

1. My results indicate that the primers provided in class functioned correctly. The small

and abundant primer segments did a good job in supporting to indicate the ends of the region to

be amplified, and in hybridizing to the complementary segments of target DNA.

2. Using the DNA standard information from the background handout, the approximate

sizes of the PCR products are about 1.2 kilobases.

3. The three basic steps of the PCR reaction are DNA melting, primer annealing, and

extension by DNA Polymerases. In DNA melting, strand separation or denaturation occurs as the

two strands of the parent DNA molecule are separated by heating the solution to 95°C for 15

seconds. Longer strands and GC-rich strands melt at higher temperatures than shorter strands and

AT-rich strands. In primer annealing, two oligonucleotide primers (one reverse and the other

forward) hybridize. The solution is cooled to about 55°C let the primers anneal to the template

DNA strand. One primer anneals to the 3` end of the template strand while the other primer

anneals to the 3` end of the complementary target strand. Then each copy will be the template in

the next cycle. Short strands can anneal more rapidly than long strands. In the extension step,

DNA synthesizes and elongates as the solution is heated to 72°C, the optimum temperature for

the DNA polymerase used in PCR.

4. Since 72°C is much hotter than most enzymes are able to function, this high

temperature will deactivate or denature most enzymes. The quality of the DNA polymerase used
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in PCR, that it is derived from thermophilic organisms where heat stability is necessary, allows it

(the Taq Polymerase) ideal for use in PCR. This polymerase elongates both primers in the

direction of the target sequence because DNA synthesis is in the 5` to 3` direction.