April 23 Polymerase Chain Reaction (PCR)

After running gel on the extraction product, it is needed to run Polymerase Chain Reaction (PCR) to amplify the extraction product. The polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. (for more information, visit Polymerase Chain Reaction.

The kind of PCR that is going to run on the extraction product was the gradient PCR with specific temperatures. The primers that were used was PHf forward primer with 5`AGA GTT TGA TCC TGG CTC AG 3` sequence and PHr reverse primer with 5‘ AAG GAG GTG ATC CAG CCG CA 3` sequence.

Materials:
Sterilized PCR tubes, marker and reagents as below

Reagents	Sample (μl)	Control (μl)	Final concentration
Template (g DNA)	1	-	10 ρg- 1 μg
Forward Primer (PAf)	1	1	0.1 μg – 1 μg
Reverse Primer (PHr)	1	1	0.1 μg – 1 μg
2 X PCR Master Mix	25	25	1 x
Nuclease Free Water	22	22	-
Total	50

Table 1 PCR Reaction Mixture

Steps	Temperature	Duration
Initial Denaturation	94°C	5 min
Denaturation	94°C	1 min
Annealing	55°C	1 min
Extension	74°C	4 min
Final Extension	74°C	10 min

Table 2 PCR Cycle Profile

Method:
1. Label three sets of tubes with A, C, D, E, F and G according to each extraction product, for example 2 stands for CC, 6 stands for S and 8 stands for K. ( in which CC is the marine bacteria from unknown sources, S is the marine bacteria from Barramundi fish and K stands for the marine bacteria from Grouper fish)
2. Start putting in the reagents according to the order in Table 1 with pipette and change the tip every time when pipette different reagent.
3. After its done, close the PCR machine and let it run for about 4 hours.
















Picture above shows the PCR Machine and the PCR tubes with samples. Gel Electrophoresis is then been run again on the PCR product. The methods of this can be acquired from the post here.

Result:




















Pictures above are the gel electrophoresis of the PCR product. The light part in each row (except for the first and last row) represents the DNA that has been amplified. Two of each samples were then picked from here and PCR purification is the next.

Conclusion:
Only tube A and tube F has the highest amount of DNA been amplified. Two tubes of each samples has been picked.