Crime Scene Samples With Gel Electrophoresis

This however, was unsuccessful, as the restriction digestion created fragments of many different lengths, resulting in smears at different lengths on the gel electrophoresis due to the restriction enzyme recognition sites being present in-between the telomeric repeat sequences, the analysis was thus

The more agarose that is used and dissolved, the firmer the gel will be. Typical concentrations used are between 0.3% to 2% (Buckingham, 2012). The concentration depends on the type of analysis needed. A higher concentration of agarose, making a stiffer gel, would be used to detect smaller DNA fragments (100-3000 bp) while a lower concentration would be used for larger fragments (5000-60,000 bp). For pieces 50,000 bp and over, pulsed field electrophoresis is used where an alternating current is applied (Buckingham, 2012). A gel comb is placed into one side of the gel mold to create holes (wells) in the gel. The gel will need to cool and solidify, about an hour. The comb is then removed, which leaves the empty wells in the gel.

Crime Scene Analysis (CSA) is a strategy that uses the behaviors manifest in an offense to predict characteristics about an offender. This type of analysis entails preserving and interpreting both physical evidence and the specific features of a crime (Turvey, 2011). In sexual offenses CSA is also likely to include data such as the relationship between the victim and the offender in the crime, the demographics of the offender, or information on how much violence transpired during the offense. Additionally, in sexual offenses whether or not the offense involved contact is also an essential component of CSA (Douglas, Ressler, Burgess, & Hartman, 1986).

The second method was to purify plasmid DNA from the E. coli culture and physcically analyze the DNA by digesting the plasmid with specific restriction endonucleases. The restriction endocucleases used were Bam HI (B), EcoRV (E), and BgIII (G). These restriction endonucleases recognize specific short DNA squences. These cut plasmids were then placed in a 1% aragrose gel to determine the band sizes. The expected phenotype for Pglo is that there will be floursenscens light produced on the LB + amp + Arab plate, where as for PFG the expected phenotype is that there will be flourcent phenotype only in LB+ Amp + IPIV. For the physical analysis Pglo will produce fragment sizes in restriction enzyme (E) 5371bp long. Enzyme (B). fragment sized 625bp, 220bp, and 4,526bp in restriction enzyme (B). Pfg will produce 2 fragments in restriction enzyme (E), one will be around 3,855bp long, and the other one will be 2,506bp long. In restriction enzyme (B) there will be only one band about 6361bp long. From restriction enzyme (G) there should be one bad around 6361bp

After completion of the restriction digest analysis, the DNA could be distinguished from one another as completion of agarose gel electrophoresis allows for the different lengths of the DNA to be observed, as GST would be shorter due to having less base pairs than GST-Cherry. Another indicator was the use of the agar plates in determining the type of DNA. In our case, our DNA had more of a clear cloudy appearance, while some others had more of a purple colored DNA. Due to this indication, it was confirmed that the different types of plasmid DNA could be differentiated based on the color of the bacteria on the agar plate. Despite the use of gel electrophoresis, our results didn’t turn out very well and weren’t very conclusive in determining the actual identity of our bacteria. It would be advantageous to retry the gel electrophoresis in order to further explore the identity of the DNA. Yet, the agar plates led to us determining what the actual identity of our sample was. Thus, in conclusion, our bacterial sample was normal GST because both of our plates, #16 and #23, contained a clear cloudy bacteria that wasn’t colored purple in comparison to the GST-cherry

The lab analysts must take extensive precautions in preventing any type of contamination because samples are limited and repeating tests is costly. At this time, the medical examiner and crime scene team are put under high pressure and must be diligent and focused when in the laboratory (Dale and Becker 139) (Roberts 48). Since errors happen because of humans and not the machines (Bertino 143). To prevent lab error, detailed guidelines for Quality Control and Quality Assurance are updated regularly (National Research Council 37). In many cases, there is an abundance of evidence that is collected and requires examination. To process all the evidence in a timely manner the forensic scientists are specialized and only process one piece in the

The world used to be a place filled with questions and answers that could not be found. As the years have gone by, evidence at a crime scene has become progressively more understood and the world is now a place filled with questions that there are answers to. To get where detectives are today, it took years of experiments, building and knowledge of technology that expanded the mind to places that are hard to believe. Technology is the basis of what it takes to find the vastest amount of information that evidence can provide. It can hold, prove and find information that in past years, could not. Due to the advances in technology, specifically through DNA, fingerprinting and lighting systems, crime scene evidence collection has grown tremendously.

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.

An important part of this is which enzymes will work in the same buffer. Using multiple restriction enzymes is necessary because without multiple enzymes it would impossible to determine the specific sequence of the DNA strand. Once you have determine which restriction enzymes you want to use, you will then set up the digest. You will need several 1.5 mL tubes (at least 7 tubes if you are using 3 enzymes). In each tube you need to add some of your DNA sample. You will then add different combination of restriction enzymes to each tube. The typical order of combination of enzymes would be 3 tubes that have one enzyme each, 3 tubes that have 2 enzymes each and one tube that has all the enzymes. It is necessary to have different combinations of the restriction enzymes so that you get a better idea of how the enzymes cut in relation to each other. Then you will add buffer, BSA, and water. Once the tubes are mixed they have to be incubated so that the restriction enzymes have time to cut the DNA into fragments. After the incubation the tubes of DNA fragments are ran through a gel electrophoresis, which allows you to visualize the results of the

As it gradually progresses to be a more improved and powerful tool in the Criminal Justice system, it should inarguably be believed that this form of forensic evidence remains as the only reliable source for providing just outcomes in a case. This technique can be discerningly used to identify criminals and exonerate wrongly-accused individuals with tremendous accuracy due to DNA’s biology to pinpoint specific suspects, it’s unique processes and the many databases it possesses. With all things considered, DNA technology has become increasingly vital to provide fairness and accuracy in the Criminal

When choosing the restriction enzymes to use, many factors are considered such as methylation of certain genes, which can influence the results of the experiment. Methylation will protect the gene from restriction enzymes, so in this experiment restriction enzymes that are not methylation sensitive are used (3). The wavelength of the UV Radiation can also have an impact on the rate of mutagenesis (4), which will be discussed more in the results. Restriction enzymes in this experiment such as Bam will cleave every six base pairs at a specific location on the target site (5). Bacteria such as E. Coli can naturally make restriction enzymes such as Eco, which is used naturally by the host cell to cut viral DNA (typically from phages) and make it non-functional in order to protect the integrity of its genome (6). In this discovery-based experiment, we will observe the changes in banding patterns of E. Coli DNA with multiple restriction endonucleases after UV induced mutation, and use these results against the control to determine if there are any changes in gene sequence by the number of base pairs in the observed DNA

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

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

To start off the experiment, 4 microtest tubes were labeled reaction tube 1 through 4. Using a micropipette, 10 microliters, ul, of Enzyme Reaction Buffer was dispensed into each of the 4 labeled reaction tubes. In reaction tubes 1 and 2, 15 ul of suspect 1’s DNA was added. Reaction tube 1 had 15 ul of Enzyme 1 added to it, and reaction tube 2 had 15 ul of Enzyme 2 added to it. 15 ul of suspect 2’s DNA as added to reaction tubes 3 and 4. In reaction tube 3, 15 ul of Enzyme 1 was added. Then, in reaction tube 4, 15 ul of Enzyme 2 was added. Finally, 2 more microtest tubes were labeled as crime scene samples. The samples contained DNA from the crime scene and each the tubes had either Enzyme 1 or Enzyme 2 added to it. The microtest tubes were then capped and tapped lightly to allow the ingredients to mix. The reaction tubes were submerged in a 37°C, ice water, for 45 minutes. Once complete, 5 ul of 10x gel loading solution was added to all 6 of the microtest tubes. Refer to Table 1 for a quick overview.

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