Dihydrofolate Reductases

The goal of the restriction digests is to be able to cut the plasmids at specific sites. This step

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 instructor split the class into two separate groups one with the plasmid lux and the other with the plasmid pUC18. Group two was assigned to test the lux plasmid. The, Eppendorf, tubes were labeled “C” for the control plasmid DNA and “lux” for the plasmid lux DNA. The two tubes were then placed into the ice bath. Using a sterile micropipette 5 uL of the lux plasmid was added to the tubes labeled “lux” or 5 uL of the control plasmid was added to the tubes labeled “C” for the control plasmid DNA. Eppendorf tubes had 70 uL of the competent cells added to them with a different transfer pipet. All the tubes were then stored in the ice bath for about fifteen minutes. Another test tube was labeled “NP”, which stands for “No Plasmid”, and 35 uL of competent cells was added to each of the test tubes labeled “NP” during the fifteen minutes. Once the fifteen minutes are up, all three tubes were placed into a preheated water bath at 37 °C for about five minutes. To both the lux

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

BoHV-1 genomic DNA was extracted using the TIANamp Genomic DNA Kit and Δ gD1 and Δ gD2 fragments were generated by PCR amplification using 2× GoTaq Master Mix under recommended setting. Each PCR product was purified using a Wizard SV Gel and PCR Clean-Up System by agarose gel electrophoresis. The purified PCR product, Δ gD1 or Δ gD2 was sequentially cloned into pET28a double digested by the corresponding restriction endonuclease, followed by transform into competent E. coli DH5α. The recombinant positive plasmid was confirmed by DNA sequencing was designated as pET28a-ΔgD1-ΔgD2.

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

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

In this lab the genetic process known as transformation will be used to make E. coli bacteria cells glow in the dark by adding GFP. Transformation is when a cell’s genotype is changed by adding exogenous DNA. This process results in the cell accepting the added genes and incorporating them into the reproduction process, creating more cells with the new gene. Genetic transformation naturally occurs in some species of bacteria, however in this lab it will be generated artificially (Bacterial Transformation). In order for transformation to take place the bacteria must be competent. This means that the bacterium needs to be ready to take in the added genetic information from its environment. These genes are absorbed through the cell membrane (Genetic Competence in Bacillus Subtilis). However, for transformation to occur, a restriction enzyme is required.

It was also conducted to learn about the process of moving genes from one organism to another with the help of a plasmid. The control group was the -pGlo with LB and ampicillin antibiotic, and the -pGlo with LB. The experimental group was the +pGlo with LB and ampicillin antibiotic, and the +pGlo with LB, ampicillin antibiotic, and arabinose. The dependent variable was the bacteria. It had the ability to change. The independent variable is the ampicillin antibiotic and pGlo. They stand alone and are not changed by other factors. The organism used was the bacteria GIVE TYPE OF BAC. and HOW WILL THE BACTERIA BE

The purpose of this lab was to observe bacterial mutagenesis of E. Coli by ultraviolet (UV) induced mutation, and observe these mutations with the use of DNA isolation techniques and gel electrophoresis vs. a control. A mutation is the changing in sequence of nucleic acids (1). When restriction endonucleases are added to DNA, the enzyme will read the certain sequence of base pairs that correlate to that enzyme of use, and then will cleave the DNA at the restriction site (2). In mutagenized DNA, the enzyme will be unable to recognize the sequence in which to cut the DNA, and this could be observed with the use of gel electrophoresis by observing the banding patterns of a control vs. the mutagenized (2). The mutagenized may also have less total

The objective of this experiment was to transform E.coli into having genes resistant for ampicillin and kanamycin by using recombinant plasmids. The three steps of the experiment include ligation, transformation, and growth on media. Restriction enzymes BamHI and HindIII splice the DNA. The recombinant plasmid is formed and combined with E.coli. Four experimental plates and four control plates were incubated at 37 degrees Celsius for 24 hours, then examined for growth. Lawns of E.coli grew on the LB plates. The

Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.

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

Two component systems are not well studied in Euryarcheota, but extremophilic methanogens and halophiles have been sequenced and researchers have found these types of systems are found in Halobacterium salinarium, Halofarax volcanii, Methanosaeta harundinacea and Methanosarcina barkeri.

Isolation of genetically marked of A. flavithermus and B. pumilus was necessary for their manipulation. Spontaneous antibiotic resistant mutants of A. flavithermus and B. pumilus were isolated by increasing the frequency of