A shuttle vector is designed to be able to propagate in two different species, for instance E. Coli and yeast. This, in turn, allows for the manipulation of DNA in a host species which might be easier to handle than the target host.
1.16 NO CLEAVAGE PRODUCTS ARE OBSERVED AFTER A DNA RESTRICTION ANALYSIS. HOW WOULD YOU TROUBLESHOOT THIS PROBLEM?
The decision tree presented in Figure 2 could be followed.
1.17 WHAT KIND OF POSITIVE/NEGATIVE CONTROLS ARE USEFUL WHEN YOU TRANSFORM A LIGATION MIX IN E. COLI?
Several strategies might be used:
1) Uncut vector alone: shows if the transformation works. If positive, many colonies would appear.
2) Cut vector alone without ligation would result in no colonies. If they appear, it means that the vector was not
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To cut DNA, all restriction enzymes make two incisions, once through each strand of the DNA double helix.
1.23 WHAT ARE RESTRICTION ENZYMES?
As suggested by the previous question, a restriction enzyme (or restriction endonuclease) is an enzyme that cuts DNA. This is usually done at or near specific recognition nucleotide sequences (restriction sites).
1.24 WHAT TYPES OF RESTRICTION ENZYMES DO YOU KNOW?
Traditionally there are 4 classifications of restriction enzymes:
i. Type I: they are complex, multisubunit enzymes with a combination restriction-and-modification activities that cut DNA at random places far from their recognition sites. They need ATP to function. ii. Type II: Unlike type I restriction enzymes, type II enzymes cut DNA at specific recognition sites (with usually 4-8 nucleotides in length) and close to them. They do not need ATP or AdoMet for their activity, but might require only Mg2+ as a cofactor. iii. Type III restriction enzymes share with type I the need ATP to function, and they are also large and multisubunit proteins. They cleave DNA about 20-30 base pairs after the recognition site. iv. Type IV enzymes only cleave modified -typically methylated- DNA.
1.25 WHAT IS
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
The product of the reaction of the A primer seems to have failed as no bands were produced apart from the terminal point of the migration which is too small to be considered as either a preintegration site or a retrovirus containing section, not only did my partner seem to have the same problem, most if not all of the submitted gels seem to have no bands for the A set of primers.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
The main goal for our experiment was to learn how to examine DNA when there is only a small
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
The novel Flight by Sherman Alexie is a story about a time traveling Indian foster kid who goes to shoot up a bank, but instead he gets transported through time and receives valuable lessons on how to deal with his main issue of abandonment. Every time he leaps into a new body the lessons get progressively difficult. Yet when he jumps into the last body, he must face the person that he blames the most, his father.
The book Flight written by Sherman Alexie is about a 15 year old part Native American
Popping pills and sticking needles down the entire length of your body sounds like a lovely way to spend free time. If you try hard enough, you might even die. Seems fun, right? Well, a matter of fact it is not. Drug abuse is a serious matter and always has been. It slips throughout your body and gives you temporary advantages but kills you over time. A great example on a more friendly level is Velocity Nine. A villain took it to gain incredible speed in attempt to beat the super hero, Flash. It was a drug that would make the weakest feel remarkably strong and let them do unimaginable things, just as a baseball player uses steroids to hit consecutive home runs. But, just as Steroids do, Velocity Nine lead to the villain 's death. He couldn’t live without it. It made his weaknesses disappear but soon after, so did his pulse.
Critique of “First Flight” The “First Flight” is an excellent short story that made pathos for the reader to portray in the life of an everyman who has to deal with exclusion and people’s bad choices. Gregory is an 18 year old who just wants to be sociable but everyone just shuts him out and doesn’t pay attention to him. He stops in a train station to warm up and is ridiculed on a false accusation of stealing a pilot uniform. W.D Valgardson perfectly shows both of the main themes.
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.
In the 1970's, scientists discovered that strands of DNA could be cut using special enzymes, which could cut out genetic combinations. DNA contains information about genes particular organisms hold. Duplicates of genes are also possible through genetic engineering and are very useful for medical purposes. Advances in technology have raised issues such as animal and human cloning. These issues have caused many different sided arguments.
How many times have an average person flown in an airplane, did people ever think what the world would be like without them? Airplanes have provided people with opportunities to go all over the world to experience different cultures and places. They also create jobs for many people which contributes to how they boost the economy. In conclusion, they appear as a more clean way to travel, helping out the Earth. The airplane was one of the most important inventions due to it creating jobs, creating easier cultural access, causes economic boosts, and created less pollution than most means of travel and also has an interesting history.