need help in finishing forensic DNA fingerprinting and PCR detection of GMO\'s F
ID: 271339 • Letter: N
Question
need help in finishing forensic DNA fingerprinting and PCR detection of GMO's
Forensic DNA Fingerprinting: 1. What are restriction endonucleases? In what type of organism were they first discovered? 2. Restriction enzymes, result in regions of the DNA called what? (hint: the ends) 3. How many microliters are in a liter? In a milliliter? How did we measure out such small volumes? How do you prevent cross contamination? What is the purpose of gel electrophoresis? What is an agarose gel? How do you make different concentrations of agarose? What was the purpose for IX TBE buffer? What is the purpose of ethidium bromide. 4. 5. How/why are the results of restriction enzymes and electrophoresis used in forensics? PCR Detection of GMO's; 1. What is a GMO? 2. How did we extract DNA from our foods? What was important about the water? And what specifically did the InstaGene matrix contain that helped protect against DNA degradation (and why)? Why did we also extract DNA from a non-GMO food? What does PCR stand for? Explain the process. What is important regarding the enzymes used? How did we make PCR specific for GM foods? What gene products did we test for? What were all relevant controls we utilized in our PCR? And what would it mean if any of the controls weren't as expected? 3. 4. 5. 6. Understand the relevance of gel electrophoresis for this application and how to interpret a gel result. Transformation of E, coli with pGLO plasmid 1. What is transformation? Which scientist's esperiment demonstrated this (and what was the esperimene)? We 2. What does it mean for a cell to be competent? How can cells be made competent? Which method did we 3. The pGLO plasmid we used was recombinant DNA. What are all the relevant components of this plasmid were transforming E. coli cells by giving it what characteristic? use? 4. 5. 6. 7. that was necessary for gaining successful results (transformation of E coli)? Media with ampicillin was used for what purpose? Media with arabinose was used for what purpose? What temperature was used to "heat-shock" the cells and why did we do this? Why did we grow cells on LB media alone?Explanation / Answer
1. Restriction endonucleases are the enzymes that cut DNA at particular restriction sites (each restriction enzyme has its specific restriction site) into fragments.
They were first discovered in Haemophilus influenza in 1970. The enzyme is called HindIII.
2. restriction enzymes results in either sticky ends or blunt ends. When the RE cuts the DNA at different sites on two strands, sticky ends are produced. These ends contain overhangs.
When an RE cuts at same position on both the strands, blunt ends are produced.
3. 1 liter = 1000 mL; 1 mL = 1000 uL.
Therefore, 1 L = 1000 x 1000 uL = 106 uL.
To measure such small volumes, we use micropipettes.
To prevent cross contamination, well sealed containers are used. The reagents are mixed in laminar air flow to minimize contamination.
4. gel electrophoresis is a technique to separate molecules such as DNA, RNA and proteins.
Agarose is a natural colloid extracted from seaweed. Agarose gels have very large pore size and are used primarily to separate very large molecules with molecular mass greater than 200 kDa. Different concentrations of agarose gel are made by using different weights of agarose powder in buffer. 1 % agarose gel is made by adding 1 gram of agarose in 100 ml TE buffer.
TBE (Tris/Borate/EDTA) buffer is used to maintain pH at 8.3 and the EDTA component of the buffer sequesters divalent cations.
Ethidium bromide is added to gel during its preparation. It intercalates the DNA. It is used to detect the presence of DNA. It fluoresces in UV light.
5. Restriction enzymes cut a specific DNA to a specific length. The DNA when run on agarose gel migrates a particular distance corresponding to its molecular weight. A molecular weight marker is also run along with the DNA in a separate well of the gel. The comparison of the DNA to the bands in marker well gives the molecular weight of the DNA.
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