CUMULATIVE LAB REPORT

Tuesday, September 6, 2016

PCR Reactions
Materials and Methods-
In the PCR reaction lab, we used:
1uL of Polymerase
1uL of 1mM DNA Template
2uL of 10mM F-Primer
2uL of 10mM R-Primer
4uL of 2.5mM NTP
10uL of x5 pH buffer
30uL of distilled water

To create a PCR reaction, use a micropipette to add all of the materials into a
small test tube. Add the materials from largest volume to smallest, ending
with the polymerase. Use the micropipette to mix the materials together in
the tube, as the polymerase will not readily disperse into the mixture.

Next, heat the mixture to 98 C to melt the hydrogen bonds between the two
strands of DNA, then cool to 64 C to allow the primers to anneal. Once this
has happened, heat to 72 C to provide the optimum temperature for
polymerase activation. This lets new strands of DNA form. Then, reheat to 98
C to melt the DNA strands and to restart the cycle. Repeat the cycle 25-40
times to allow a significant amount of DNA to form.
Results-
While there is no difference visible to the human eye at the end of this lab,
the results of the gel electrophoresis lab indicate that the PCR process was
successful.
Conclusion-
The evidence produced in the next lab when we preform gel electrophoresis
indicates that the PCR reaction did occur as expected and replicated the
target strand of DNA.

Sunday, September 11, 2016

Gel Electrophoresis
Materials and Methods-
In the Electrophoresis lab we used:
The product of the PCR reaction
UV Fluorescent dye
Agar Gel
Electric Current
Conductive Solution

To conduct Gel Electrophoresis, take the product of the PCR reaction and add
it to the wells in the Agar gel. Next, add the dye to the PCR product in the
wells. Submerge the Agar in the conductive solution and run an electrical
current through it with the positive end opposite the wells. Since DNA has a
negative charge, the positive charge will pull the DNA out of the wells and
through the Agar, where the smaller strands will smooth faster. Wait until an
appropriate amount of time has passed, in our lab it was 40 minutes, and
then remove the current. Observe the gel under UV light to locate the target
region, and then carefully remove it.
Fig. 1

The agar block after electrophoresis, note the target region with the more
intense bands.
Results-
The intense bands of DNA fragments at the same distance down the gel
indicate that the target DNA was replicated by the PCR, and the fainter lines
indicate that there were other other sequences replicated as well, possibly
by partial attachment by the primers.
Conclusions-
The electric current drew the DNA fragments through the gel where the
larger were caught in the fiber matrix earlier than the smaller ones.
Fragments of similar size stopped near the same spot, resulting in bands that
show up under the UV light.
 Thursday, September 15, 2016
Gel Extraction
Materials and Methods-
In the Gel Extraction lab we used:
The product of Gel Electrophoresis
500uL Binding Buffer
Silica Column
Centrifuge
500uL ethanol solution

To extract the DNA from the Gel, add 500uL of the binding buffer and
incubate until the gel is fully dissolved. Add this solution to the silica column
and run it in the centrifuge at 21,000 G for one minute. When this is done,
discard the flow through. After this, add 500uL of ethanol solution to the
column and run it in the centrifuge, once again at 21,000 G for one minute
and discard the flow through. Finally, wash the column with a TE buffer in the
centrifuge at 21,000 G for one minute and collect the remaining solution.
Results- While not apparent to the naked eye, the DNA has been removed
from the gel and purified by the repeated washings and runs in the
centrifuge, allowing it to be used in the next lab, Restriction Cloning.
Conclusions- This process worked because of the order in which we washed
the DNA. The binding buffer prevented water from interacting with the DNA,
which allowed the DNA to bind to the column. Then, the ethanol washed of
any impurities without dissolving the DNA, finally, the TE buffer chelated any
magnesium that would have caused the DNA to react with anything else in
the mixture, thus purifying the solution.

Thursday, September 29, 2016

Restriction Cloning
Materials and methods- In this lab we will need:
Plasmids
DNA product from Gel extraction
Restriction enzymes ( Nde1 and BamH1)
Buffer solution

Put 19.5uL of the combined solution of plasmids and DNA into a test tube,
and combine it with1.5uL Nde1, 1.5uL BamH1, and 2.5uL buffer solution. Mix
these with the pipette and incubate at 37 C for one hour. Add the solution to
a culture of DH5a ecoli and let the ecoli reproduce.
Results- This process will insert the DNA into the plasmids, which will then be
taken up by the ecoli and reproduced as the bacteria reproduce.
Conclusions-For this process to have worked, the restriction enzymes must
have had to cut the plasmid at the appropriate spot to leave sticky ends
where the DNA strand could bond.