IntroductionPolymerase experiment was M13Foward (5′-CCAGGGTTTTCCCAGTCACG-3′) and M13Reverse (5′-TCACACAGGAAACAGCTATG-3′).

 IntroductionPolymerase chain reaction (PCR)
is technique in biochemistry to amplify a single or few copies of DNA.  The invention of PCR was discovered in 1993
by Kart Mullis due to it he won a Nobel prize in chemistry.                Plasmid is a small circular DNA
of a bacterium which is independent form chromosomal DNA and it can replicate
itself. Bacterium can be manipulated by inserting a foreign DNA in to the
section of the plasmid. The three plasmids used for this PCR reaction are
cSox1, cWnt1 and cMyod. It’s possible to amplify a certain of a plasmid since
the primer binds to the vector they can used regardless of the insert sequence
therefore they are called “universal primers”.  Performing PCR requires 3 steps.
The first one is denaturation which is specifically for breaking the hydrogen
bonds between the double stranded DNA molecules, this is by applying some heat
with a temperature of 95°C for about 15 seconds. After separation of the double
strand, the second process of PCR is annealing which binds the primers onto
strands of the DNA by cooling the heated solution to a temperature around 57°C
for about 15 seconds. The primers used in this experiment was M13Foward (5′-CCAGGGTTTTCCCAGTCACG-3′) and M13Reverse (5′-TCACACAGGAAACAGCTATG-3′). Primer
attaches itself into the opposite strand of the vectors DNA by doing its
possible to amplify the selected area of the DNA. The step that ends the
process is called extension, Taq polymerase can withstand high temperatures that
it is ideal for PCR. used nucleotides to the correct base pair on the DNA. New
Double stranded DNA segment is created by Taq polymerase by building each
single strand of DNA marked by the primer. The polymerase begins DNA synthesis
on the primers and elongate in the 5′ to 3′ direction on both ends so the
cooled solution is heated for 1min to a temperature of 72°C. The result of the
PCR produces exponential amplification so at 36 cycle there is 68 billion
copies of the selected DNA.Gel electrophoresis is used to
separate the DNA sample as it is negatively charged thus applying an electric
field will allow DNA to move through the gel, consequently smaller DNA fragment
moves further away whereas larger DNA fragment travels less.     MethodIn this experiment contamination
needs to be avoided since PCR is a highly sensitive method so wearing glove is
necessity for this experiment.Firstly, PCR tube was labelled as
1, 2 and 3. In each tube 9.5µl of water, 12.5µl 2X Biomix red, 1 of M13F and M13R
primers was added. 1µl of the appropriate plasmid DNA (1,2,3) where each PCR
tube was containing only one type of plasmid)Secondly, A fresh tube was
labelled as N, the 1 µl plasmid DNA was replaced by 1µl of water. Thus the
negative control contained 10.5µl of water,12.5 µl 2X BioMix Red (Taq
polymerase, dNTPs, buffer),1 µM13R primer (50 µM), 1 µl M13F primer (50 µM) The tubes were then placed in the
PCR machine and the following programme was running. There was initial
denaturation for 1 minute at 95°C then 25 cycles of denaturation taking place
15 seconds at 95°C, primer annealing of 15 seconds also at 57°C and extension
of 1 minute at 720C. Finally, the final extension of primer took 5 minutes at
72°C The gel solution is poured onto
the electrophoresis tray and enough buffer was added to cover the gel. 12 ?l of DNA molecular weight marker of 1kb ladder
is loaded onto the first well, pipette 8 ?l
of each PCR samples onto gel in the following order of N,1,2 and 3. Electrodes
are placed on each end, cathode being near the wells and anode being further
away from the sample, the Gel was run at 120V until the red dye was about 2cm
from the bottom. Ethidium bromide is added to the solution so when the UV hits
the sample the DNA fragments becomes fluorescent.   Results
and Discussion                                                  I decided to use linear regression to estimate the
kilobase as some of the fragments are larger than the ladder due to this
migration from well to fragments midpoint was used to calculate kb.                    Above is the graph between log10(kb) against
migration although the R2 of the linear regression (R2=0.9886)
is lower compared to 5-degree polynomial regression (R2=1), due to
the error caused by the fragment size height compared to the ladder, the linear
regression model yielded a better result.     
 

Migration 1(mm)

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                Log10(kb)

Kb

27

0.347

2.22331

48.5

-0.36465

0.431867

Migration 2(mm)

 

 

24

0.4463

2.794474

55

-0.5798

0.263148

Migration 3(mm)

 

 

24

0.4463

2.794474

38

-0.0171

0.961391

47

-0.315

0.484172

55

-0.5798

0.263148

 The result of the gel electrophoresis shows different
size fragment due to the three plasmids used as template, cSox1, cWnt1 and
cMyoD. The negative control is used to identify contamination in the resulting
experiment, since there is no fragment in the coulomb his is to be expected.
The intensity of the band is due the abundance of the DNA fragments, this is
the result due the PCR amplifying the insert. 
The amplified DNA fragment was much thicker which may cause it to
overlap with some fragment that are bit bigger or smaller. On the PCR
product 1, band on 0.431867 kb is brighter than the band on 2.22331 kb, this shows that the insert is
amplified by a lager factor and since the insert is ?0.6
kb (600 base pairs) so it is cWnt1 plasmid DNA. Whereas PCR product 2, band on 0.263148 kb is more fluorescent than the band
on 2.794474 kb, so
the insert at ?0.3kb (300 base pairs) has been amplified by PCR which
shows that this is cSox1 plasmid DNA. On PCR product 3, there is an unexpected result as it was
expected to be two bands but the PCR process amplified the correct insert 0.961391
kb which is roughly 1kb
(1000 base pairs) this show that this is plasmid DNA cMyoD. But pBluescript is expected to be ?3 kb long but there are few fragments in 0.3 kb and 6 kb,
this could be due to various such as primer binding to other site where the it
only few cycle of amplification occurred, or nucleotide interaction in the
solution.  ConclusionPCR can be used to amplify a wanted DNA segment, for the
case of this experiment we can amplify a certain wanted DNA fragment as an
insert which can be used to modify the plasmid of bacteria. This process is
used to create insulin, which helps regulate blood glucose level, by adding a
certain segment of human DNA which contributes to the production of insulin to
the bacterium’s plasmid and let bacteria grow into colonies to produce insulin.
By using linear regression, the kb ladder was modelled
with respect to the migration of fragment from well, this was because of the
compressed spacing and faded band causing the band’s position relative to the
kb ladder to be in accurate. Using the modelled linear regression line the PCR
product 1,2 and 3 was predicted which gave an fairly accurate result and help
identify the size of the figments as well as the different plasmid DNAs using
the size of the insert.The result consist of various error as some of the band
was very faded and the space between were very compressed resulting in being
hard to identify the correct base pairs. The spacing between the bands can be
increased by increasing the electric field between the cathode and anode, by
rising the voltage between them.