Restriction Enzymes Lab Report

 By restriction enzymes then amplified by polymerase chain reaction to make many to millions of copies of a single fragment.

(PCR), which isolates small fragments of DNA that have a high degree of variability from

We were using a restrictive enzyme to cut the DNA into smaller fragments. For the restriction digest we pipet 4 micrometers of enzyme mix into the bottom of each of our colored tubes making sure to use a new tip for each sample. Next, we capped the tubes and mixed the contents by flicking the tube a little bit with our fingers. After we mixed the contents, we tapped the tube to make sure all the liquid would go to the bottom of the tube. Then, we put the colored tubes in the heating blocks and we let them incubate for 35 minutes at 37 degrees Celsius.

The goal of the restriction digests is to be able to cut the plasmids at specific sites. This step

Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific

PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. Steps of the PCR cycle are shown in below figure.

Lab Report on pGAL Transformation In order to understand this lab the student first needs to understand how recombinant DNA is formed. To begin, the student extracts a plasmid, which is a circular strand of DNA found within bacterial cells, from the bacteria. Restriction enzymes begin to cut the plasmid at certain sequences of nitrogenous bases.

Let sit at room temperature for 2.5 minutes 9. You will start to see a cloud film develop into the layers of solution. (This is your DNA) 10. Gently spool the DNA onto a glass rod, and dissolve into 1000µ of 1xTE buffer. 11.

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

A basic method in which we get specific genes integrated with another organism’s chromosome is as follows: Isolate the DNA from which selected gene is to be taken from and treat it with enzymes that will cut out that specific gene. These genes are then inserted into bacteria and grown into colonies being

Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA

DNA fingerprinting is a technique that is used to determine how likely it is whether genetic material came from a specific person or family group. Since 99% of human DNA is identical, that means that it is only 1% of our DNA which is different, and it is that 1% that we look at when we are attempting to determine the origin of a DNA sample.

To sequence DNA it needs to be amplified first and this can be done in two ways; The Polymerase Chain reaction (PCR) and Cloning. Cloning isolates the DNA fragments from restriction enzymes and then combines the fragments with the vector. Once united with the vector the DNA fragments are then able to replicate to provide an unlimited amount of DNA for the experiment. PCR allows DNA to be multiplied millions of times in a couple of hours, PCR is used because it’s highly specific, easily automated and can amplify very small samples of DNA. The technique of PCR has had a huge effect on genetic disease diagnosis. To sequence the DNA, Gel Electrophoresis is used as it separates DNA fragments according to their size by running an electric current through the gel (Boehm). Mapping and sequencing the genome of organisms has allowed tests to be conducted, for example; gene tests, this is where individual genes or short lengths of DNA or RNA are tested for and Chromosome tests; this is where whole chromosomes or long lengths of DNA are tested for (National Human Genome Research Institute, National Institutes of Health 2014c).

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double