Restriction enzyme

MMR Report 1.3 Restriction Enzyme, Alkaline phosphatase Digestion 
and Gel Electrophoresis By Naga Srilekha Somu Chemistry - 429 Spring 2016 Western Illinois University Materials and Equipment: Pure plasmid pET28a, amplified 2-alcohol dehydrogenase gene (a PCR product), 10x bovine serum albumin, 10x neutralization buffer, EcoRI, nuclease free water, pET28a plasmid digested with EcoRI, calf intestinal alkaline phosphatase, agarose gel (1% agarose + 0.3μL ethidium bromide), 1x TAE buffer

the suspect of the crime? Why or why not? The DNA found on the crime scene matches the one from suspect 3 through same number of cuts and size of the banded fragments which are both identical. This shows that two samples of DNA have identical restriction sites that produced identical patterns. When also calculating the number of base pairs of all samples, we find that there is a 97.76% match with Suspect 3's DNA with the crime scene DNA

The DNA from the two individuals who were linked to the skeleton: Missing person #1 and Missing person #2 were then analyzed. The first part in finding the DNA was DNA digestion with restriction enzymes. That’s when 4 DNA samples, the DNA of person #1 and #2 that were treated with two different restriction enzymes, were taken and then incubated in a 45C water bath for 15 minutes. After that 5 microliters of 10X gel loading solution were added to the DNA samples to stop the reactions. The samples

1. The reagents needed to run a restriction digest lambda with the enzyme HindIII will include lambda DNA, 10X restriction Buffer, HindIII and water. 2. The following would be added to an Eppendorf tube: ~5uL lambda DNA(0.5ug/uL) ~5uL 10X restriction buffer ~5uL HindIII ~35uL water This amount would give you a final reaction volume of 50uL. The reaction is mixed and centrifuged to draw the reaction to the bottom of the tube. The reaction was then incubated in a dry bath at 37°C for 1 hour.

(pH 8.4) 0.8 µl MgCl2, 0.7 µl dNTPs, 1 µl of each primer and 0.5 U Taq DNA polymerase. Then 5µl of products were incubated with 0.4U of Taq1 restriction enzyme at 65 °C for 48 h. Finally the products of the Taq1B digestion were electrophoresed on 1.5% agarose gel. For -629C/A polymorphism, 5µl of PCR products were incubated with 0.4U of Van91I restriction enzyme at 37 °C for 48 h. The thermocycler conditions after optimizing the technique for -629 C/A were: initial denaturation at 96°C for 5 min followed

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

Increasing enzyme concentration increases reaction speed. This is due to the maximum velocity level in which the enzyme is able to be active. As the enzyme concentration increases it compensates for the amount of restriction factors such as temperature, pH and substrate levels; all of which increases their effectiveness due to an imbalance in substrate (Eed, 2013). This was shown using an increased Pyrosphosphatase concentration throughout the experiments. As shown in figure 1, the enzyme hydrolysis

The clarified supernatant was directly purified using a Ni-NTA Spin Column according to instructions supplied by the manufacturer (QIAGEN). Briefly, after washing the column with wash buffer1 (50 mM NaH2PO4 pH 7.5), the supernatant was loaded onto the column, and the column was washed with wash buffer 2 (50 mM NaH2PO4 pH 7.5, 500 mM NaCl, 0.025 Triton X-100 and 20 mM imidazole). The integrase protein was eluted from the column using elution buffer (50 mM NaH2PO4 pH 7.5, 500 mM NaCl, 0.025 Triton

Enzymes are catalytic proteins that accelerate the rate of biological reactions while experiencing no permanent chemical modification as a result of their participation in a reaction. In order to initiate a reaction from a reactant called a substrate to a product, a certain amount of energy, otherwise known as the activation energy, is required. An enzyme functions by lowering the required activation energy (which is usually provided by heat), thus, expediting the reaction. Many chemical reactions

ensure amplification (Figure 4). The PCR fragment was purified using the Quick-Start Protocol for the QIAquick PCR Purification Kit. After the purification of the PCR fragment, a digest of the M. Xanthus PCR fragment into the PET45 plasmid using enzymes KPN1 and HINDIII was performed.