Dihydrofolate Reductases

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

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

At the same time, bacterial colonies are needed to be placed into PCR, and use photograph of gel to determine the size of tetracycline resistance genes to distinguish the bacteria. It is relevant to research 1 because serial dilution is the first step to get colonies' samples that is further being used to distinguish plasmids. It is also relevant to research 2 because serial dilution is also the first step to count the frequency of tetracycline resistant. Week 2: Experiment Part B By placing the bacteria into the PCR machine, much more copies of genes would be made to make them visible under electrophoresis, which can then be compared with control ladder to distinguish the kind of bacteria.

This allows for the assumption that the mutation in the unknown strain #175 was either the metA+ gene or metB+ gene. The lack of intermediate between metA+ gene and metB+ gene, made it impossible to pinpoint the exact location of the mutation using the biochemical pathway analysis. This prompted the second portion of the experiment in which the unknown auxotroph was transformed using metA+ gene and metB+ gene ,which were then plated on both MM and LB plates containing chloramphenicol in order to identify the specific gene that is mutated in the unknown strain. On the minimal media, the only growth exhibited was from the one rescued with metB+ (figure 2), indicating gene B was the one that was

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

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

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

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.

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

Any remaining histidine media was washed off of the C. albicans culture, and then the C. albicans culture was centrifuged. The C. albicans culture was washed twice with SD-min. A seven 10 fold serial dilution for the C. albicans culture was performed using SD-min. A portion of the diluted culture was plated onto each YPD plate. (There were three YPD plates, which were 10-5, 10-6, and 10-7 dilution.)

In biochemical test, which is base on the hydrolytic catalytic enzyme that show their main similarities between Halo bacterium Salinarum and Haloferax volcanii that focus from its result which show to be as negative that is turn out to be as gelatin and whether it can able to substantiate but it’s not possible as is too difficult to melt it. Concern from its illustration that it is been demonstrate on how negative effect is focus also from casein hydrolysis and from Tween 80 and Tween 20. But in Haloferax mediterranei it show the main difference that its result that show to be as positive but is also turn out on gelatin and how gelatin and casein can able to transform into amino acids that is determine from its source like carbon and other

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