Bacteri Bacteria And The Test Tubes

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

The purpose of the DNA restriction and electrophoresis lab was to first become familiar with the properties of restriction enzymes and discover that they, along with agarose gel electrophoresis, are used to characterize DNA molecules. Restriction enzymes are used to make cuts or join together DNA fragments. The cuts result in either staggered cleavage, or blunt cleavage. Staggered cleavage results when the breaks are offset. This cleavage usually results in sticky end being produced because for each DNA end that is produced there are extensions with unpaired nucleotide bases. Blunt cleavage results when the breaks in the phosphodiester bonds are right across from each other.

In this experiment, host NM554, a particular strain of E. coli, was used to cultivate human genes (Dolf, 2013). Through the use of cosmids, plasmids that carry the cos gene, DNA fragments were introduced into the E. coli and packaged into phage particles (McClean, 1998). Pst I is a restriction endonuclease, an enzyme that cuts DNA at restriction sites (Restriction endonuclease). The Pst I digest of human DNA in this study produced the DNA fragments that were examined. The dideoxynucleotide chain-reaction procedure, also known as Sanger sequencing, is the process of lengthening DNA using DNA polymerase to add on deoxynucleotides until a dideoxynucleotide is added on randomly (Rogers). Fluorescence in situ hybridization (represented by the acronym

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

The recommendations before starting the lab are to conduct a practice experiment with inserting the DNA with the micropipette. This decreases the chance of rupturing within the gel, and if this is to happen inaccurate results will be displayed. To begin the experiment, insert the gel into the electric chamber filled with THE solution so that it covers the agarose gel. Once the agarose gel is in place, take the Missouri DNA and extract from the sample tube with the micropipette and insert it into one of the wells. Repeat this process with each tube, but after inserting a DNA replace the disposable plastic tube with a new tube, so no inaccurate results will occur. After all six wells are filled, enclose the chamber and initiate the chamber with

Each lab bench will make, and run one gel electrophoresis per table. Once the gel is ready to be loaded, load five microliters of PCR DNA ladder into the first well, as a standard. This should be found in a tube in and ice bucket. Next add two microliters of 6x loading dye into the six sample tubes. The dye should be mixed in thoroughly by gently pipetting up and down after adding the dye. Following that you should load fifteen microliters of each sample into the following six wells. Since lane one will have the DNA ladder lane two starts the samples using the orange tube, then the blue, yellow, red, green, and pink tubes go into lanes three, four, five, six, and seven respectively. Once all the samples are loaded turn on the electrophoresis machine, and wait until the bromophenol blue tracking dye has migrated at least half the length of the gel. Lastly using gloves carefully remove the gel and carry it to the UV light box to view, and photograph the gel (Hass C., Woodward D., and Ward A., 2010.).

Bacteria are the smallest living organisms, they are prokaryotic and have a simple cell structure. They do not contain a nucleus, and are unicellular. Bacteria are the most abundant microorganism. They are among the earliest forms of life that appeared on earth billions of years ago. They can grow in different temperatures and many exist naturally in our human flora, such as staphylococci. Growth within different temperatures can range from 0 degrees Celsius such as psychrotrophs, to hyperthermophiles who grown best above 70 degrees Celsius (Ritchey, Exercise 16). Bacteria have many shapes, size, and multicell arrangements. The main bacteria shapes are cocci (spherical or ovoid shaped), bacilli (straight rods), coccobacillus (short rods), fusiform bacillus (rod shaped bacilli with tapered ends), vibrio (curved rods), spirillum (single spiral bacteria), and spirochete (if it is flexible and undulating). Bacteria can form multicell arrangements, such as diplococcus (two-cell arrangements) (. Bacteria are usually named for their shape or multicell arrangement, but cannot be truly identified by just those characteristics (Strelkauskas, Edwards, Fanhert, Pryor, 2016). Bacteria can be classified by the way they stain. They are clear under a microscope and require staining to be seen. To stain a bacteria, one takes a dye to color the cells. There are basic dyes that contain

Then the sample is prepared using glycerol, which makes the sample denser allowing it remains in the wells. Since DNA is clear a dye or fluorescent agent can be added before or after the gel has been run. A micropipette is used to load the wells with the sample. Also, a DNA “ladder” is placed in the left most well and is used to identify the approximate molecular weights of the unknown DNA samples.

The product of the restriction enzyme digestion of plasmid DNA was analysed visually on agarose electrophoresis. The identity of the plasmid fragment in plasmid 1 at 1122 bp is established to be Integrin. The identity of the plasmid fragment in plasmid 2 at 501 bp is determined to be VEGF.

Add exactly two microliters of each sample and standard to their corresponding marks on the plate. Place the bottom centimeter of the plate into a glass jar with approximately 500 microliters of ethyl acetate. Allow the ethyl acetate to be absorbed up the plate until three centimeters from the top of the plate. View the plate under a UV light and mark the movement of each analyte with pencil. If the analyte is in the same position as the standard BMK, place the test tube’s solution

Gel electrophoresis is a commonly used laboratory technique employed in biochemistry and molecular biology (ARBL, 2000). The two most conventional types of gels used for DNA electrophoresis are agarose and polyacrylamide (PA). The two substances differ in factors such as resolving power and in the difficulty of setting up and handling them (ARBL, 2000). In comparison to the polyacrylamide, agarose gels are used more commonly as it may also be refrigerated and re-used, and runs horizontally (Reina, 2014). Gel electrophoresis through an agarose channel is used to identify, quantify and purify nucleic acid components (Life Technologies, 2015). The samples of DNA are loaded into wells of agarose gel which is then subjected to an electric current,

Once the pellet had formed I carefully poured out the liquid that was above it leaving just a little bit of liquid in the tube with the pallet. Then taking a 200 microliter PCR tube, using a micropipette with a fresh tip, I first added 100 microliters of Chelex solution, which is used for DNA extraction when preparing for PCR, and then 30 microliters of my cell suspension which was in the microcentrifuge tube. That PCR tube was placed in the thermal cycler for 10 minutes at 99 degrees celsius to break down the cell so DNA can be released. Once the 10 minutes was up the tube was shaken up in the vortex for 5 seconds. The tube was then put in the microcentrifuge for 90 seconds to form another pellet. Once the 90 seconds was up I took the micropipette again with a new tip and transferred 30 microliters of the liquid on top of the

Place test tubes 1 and 2 into the ice bath, test tubes 3 and 4 into the RT (room temperate) water bath, test tubes 5 and 6 into the 35oC water bath, test tubes 7 and 8 into the 50oC water bath, test tubes 9 and 10 into the 60oC water bath and test tubes 11 and 12 into the 90oC – 95oC water bath. Ensure the hot test tubes are held with metal tongs to avoid

7.When water bath is ready, put each test tube into the water bath. Wait 5 minutes.

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