DNA isolation and amplification of ansA1 and ansA3 genes Good quality genomic DNA was isolated

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).

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

In this section thermal stability of the purified enzyme was studied at pH 5.5 and all optimum conditions of enzyme and substrate concentration. The enzyme solution was preheated at different temperature 40°C, 50°C, 60°Cand 70°C for various time periods from 10 to 60 minutes. After each heat period, the enzyme was rapidly cooled in an ice bath. The activity of pectinase was determined by adding the substrate and carrying out the enzymatic reaction under optimum reaction conditions.

Blood samples were obtained from all family members. Genomic DNA was extracted from 100-μl samples of peripheral blood mononuclear cells incubated with proteinase K in lysis buffer, followed by phenol/chloroform extraction and ethanol precipitation. Finally, the pellet was dissolved in 30 μl sterile water and stored at -20°C until further tested. To determine mutation, specific primer sets within the APC gene were designed (primer sequences upon request). Two microliters of DNA were used to set up 25-μL PCR reactions by using the Perfect Taq plus MasterMix (5 prime GmbH, Hamburg, Germany). The amplification reaction was carried out under the following conditions; pre-incubation at 95°C for 3 min, followed by 35 cycles comprising denaturation at 95°C for 30s, primer annealing at 55°C for 30s, and extension at 72°C for 1min, and concluded by a final extension at 72°C for 5 min. Upon electrophoresis in 2.0% agarose gel stained with EtBr, the amplification products were visualized under UV

Cellulase. Cellulase from Aspergillus niger (Cat: C1184, Sigma-Aldrich) was used in same concentration as pronase E i.e. 100 enzyme units (U) per 200 µl. The activity for cellulase was marked as 1.3 units/ mg solid and hence 1.92 grams of cellulase powder was added to 5 ml Tris (0.05 M, pH 7.4) to get desired stock solution concentration of 500 U/ml.

2. McClean, Phillip. (1997). Cloning and molecular analysis of genes: Polymerase chain reaction (or PCR). Retrieved on December 6, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/ clone9.htm. 3.

Hypothesis: Ψ-mRNA, synthesized via in vitro transcription, will have enhanced stability and decreased immunogenic properties. Restriction enzymes will be used to clone the NT-3 gene (NTF3) into the pDNA expression vector known as pDNA3.1. DNA polymerase, deoxynucleotide triphosphates (dNTPs), a forward primer containing a T7 promoter, a reverse primer containing a 75 thymine tail, and the pDNA3.1 vector will be combined in a polymerase chain reaction (PCR) tube. Standard PCR techniques

BglA was remain active even in the existence of 600 mM glucose. However, BglA activity was gradually repressed with the increase of glucose concentration, and Ki value for glucose was found to be 1200 mM as shown in figure 4b. Organic solvents have proven an obvious constrain for the BglA activity, enzyme was completely inactive by adding 40% (v/v) methanol, ethanol, n-butanol and isopropanol whereas, 40% (v/v) acetone, ethyl acetate and acetonitrile strongly repressed BglA activity up to 18%, 38%, 50%, respectively.

The results from this experiment are inconclusive as the 1% agarose gel using 142S primers did not display any bands (data not shown). The 142S transgene should be present in the 142S line only. In the control gel (Fig 3), there are bands in every lane; actin is used as the loading control, showing the PCR and gels were run according to standard procedures.

Background & Objectives: Digestive enzymes are present throughout the digestive system and are used to breakdown various macromolecules obtained from food. An enzymes activity is dependent upon the temperature and pH at which it is present. Enzymes work optimally at specific temperature and pH levels. The purpose of this lab is to examine various enzymes to determine which substrates they digest, and at which pH they work optimally.

Numerous standard laboratory techniques and instrumentation was used to obtain the forthcoming results. The ASAP1 expression plasmids were created by standard molecular biology methods, and then verified through cloned fragment sequencing. A plasmid was used for multiple reasons, the most prominent being that plasmids are very easy to manipulate to express a desired genetic element. In addition, they are very stable as well as self-replicating once initially constructed. HEK293A cells were used in experimentation and transfected with the ASAP1 plasmid. The HEK293A cells, which are very popular for transfection, were chosen as they are easy to reproduce, maintain, and have a high efficiency of transfection. HEK293A cells also have minimal processes that need to be accomplished in order to preform voltage-clamp experimentation. Transfection is simply the process by which

Nevertheless, the results of these genetic studies have been rather disappointing and new investigations are requested in order to significantly increase the chances of finding strong gene/phenotype

Awan et al., (2011) reported that Aspergillus niger is a potent producer of many industrially important enzymes and may genetically be improved by exposure to gamma rays. The mutants recovered after treatment by gamma rays were found to be effective producers of enzymes (Awan et al., 2011). Mutagenesis of Aspergillus niger by using chemicals has been reported earlier to improve many industrially important enzymes and other products. The cells of Aspergillus niger were subjected to mutagenesis by ultraviolet irradiation, resulting in 45.4% activity of cellulase (Junwei and Shuyun, 1988). Non

Certain industrially used microorganisms have been genetically modified to increase productivity, the desired activity and not to produce undesirable side effects. Often enzymes do not have the desired properties for an industrial application. E.g extreme thermo stability or overproduction of the enzyme. Protein engineering is used to improve commercially available enzyme to a better industrial catalyst. Several enzymes have already been engineered to function better in industrial processes. These include proteinases, lipases, cellulases and few amylases.

The first strand cDNA was synthesized using 5µg of total RNA. The RNA and primer mix was incubated at 65°C for 15 min and then the tube was placed on ice for 5 min for chilling. Subsequently, 200 ng of gene specific primer, 5µl of RT-Buffer (10x), 0.25µl of RNasin (40U/ µl), 2 µl of dNTP mix (2.5 mM each), 5µl of DTT (0.1M), 2 µl of MMuLV RT (20U/µl) was added and the final volume 50 µl of reaction mixture was prepared using DEPC water. Reaction mixture was incubated at 42°C for 1 h and the reaction was terminated by heating at 94°C for 5 min followed by snap-chill on ice. The first strand was used for the PCR with gene specific primer for the second strand synthesis followed by cDNA. Further, reverse transcriptase - PCR (RT-PCR) was performed