Circadian Clock Biological Lab

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 variables in this lab were different than the average science experiment. Instead of affecting the experiment to prompt different results, we just had F1 generation plants produce offspring so that we could study their specific traits. By looking at the variables, we can determine if they fit the Mendelian ratio and see if genes are linked on a chromosome.

Experiment 4 & 5: PCR did not work for my obtained DNA as there was no evidence of movement of the DNA. PCR did work for lab numbers 33 and 12. The size of my fragment could not be determined and cannot compare to the size that was expected. The negative control was clean as there was no traces of movements.

Also we will sequence the PCR product when we see a shortened product in the exon-skipping group. Primers therefore requested will be developed. These primers will be designed to attach to exon 3 and exon 6. Western blots will be performed after successful exon-skipping is seen in RT-PCR. An antibody will be designed against Notch3 for a sequence that is not

Bimonthly, a random sample of 5 mice will be taken and analyzed for levels of expression of peripheral clock genes in the form of PER proteins in the cytoplasm of cells [5]. The relative levels of the proteins in the cytoplasm will be compared between the three test conditions and analyzed to determine the effects of a high fat diet on the sleep cycle. Each sample of mice will be euthanized and a sample of Epididymal adipose tissue will be taken [6]. Proteins and primary RNA transcript will be extracted from adipose tissue via a TRIzol extraction kit following the protocol published by The Institute for Genomic Research [6]. The sample taken from these mice will have varying levels of PER and CLOCK protein. To help further quantify the relative amounts of these proteins in the extracted sample an ELISA assay will be used. [7]. An ELISA assay will require the purified sample, an antibody specific to the CLOCK protein, and a substrate specific to the antibody [7]. The CLOCK specific antibody will react with the relative amounts of protein extracted from the sample, which, by the addition of a substrate, will be translated into a measurable signal. Once completed, the signal will be analyzed by a plate reader, which uses a program to measure the intensities between the various samples and quantifies the

. In the first step, denaturation, the DNA is incubated at 93-95C for 30 seconds. This breaks the hydrogen bonds between the nucleotide base pairs and separates the two strands of DNA. In the second step, annealing, the reaction is incubated at 45-65C for 45 seconds to 1 minute; the presence of excess primers allows the complementary primers to hybridize to target DNA. The third step, primer extension, is conducted at 72C from 15 seconds to 1 minute and involves DNA synthesis, in which two new daughter strands complement to the original single

Introduction Chemistry can be divided into the study of structures, equilibriums and rates. In a chemical reaction, a system is said to be at equilibrium if there is no change with time in any of the measurable properties. However the fact that there is no measurable change does not mean it is static; just perhaps very slow (Sposito, 1994). At equilibrium the flow of atoms or molecules in one direction is equal to the opposite (Nevers, 2012). Similarly the concentration of all substances does not change over time.

Much can be learned from studying an organisms DNA. The first step to doing this is extracting DNA from cells. In this experiment, you will isolate DNA from the cells of fruit. Materials (1) 10 mL Graduated Cylinder(2) 100 mL Beakers15 cm Cheesecloth1 Resealable Bag1 Rubber Band (Large. Contains latex pleasewear gloves when handling if you have a latex allergy).Standing Test TubeWooden Stir StickFresh, Soft Fruit (e.g., Grapes, Strawberries, Banana, etc.) ScissorsDNA Extraction SolutionIce Cold EthanolYou Must ProvideContains sodium chloride, detergent and waterFor ice cold ethanol, store in the freezer 60 minutes before use. Procedure If you have not done so, prepare the ethanol by placing it in a freezer for approximately 60 minutes.

This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and

The mixed PCR product was loaded into the gel, and the electrophoresing began in order to separate genomic DNA. The second part of the experiment PCR reaction was DNA sequencing; which was processed by the DNA Learning Center in Cold Spring Harbor, NY. The purpose of sequencing DNA was to determine the exact sequence of nucleotide in a given piece of DNA. The third portion was processed by the lab assistance. The purpose of the third method DNA repair was sequencing to test the effect of UV-light on two different strains of yeast for identifying cell with a mutation required for NER. The fourth process of PCR is DNA purification and RFLP. In DNA purification, QIAquick PCR Purification Kit was used to purify amplicons from other contaminants following the steps in material and methods. For RFLP analysis, the purified amplicons were cut using JOSH04 and JOSH05 PCR reaction with two different restriction enzymes. MsaI and RsaI were used to cut mtDNA control regions from the purified DNA. The fifth method of PCR was running the RFPL sample on gel electrophoresis. Most people within a population have many single nucleotide differences within their genomes. These differences are often

Gene-specific primer pairs were designed using NCBI Gene database and primer design web tool (http://www.ncbi.nlm.nih.gov). The web tool generates a series of primer sets that fit the parameters defined by users. These parameters include size of the final product and the primers, whether primers span the junctions between exons, GC contents of the primer etc. For each isoform

When temperature is lowered to 42°C, primers anneal to complimentary target RNA. After multiple rounds, amplification of cDNA can be quantified and analyzed. The amount of amplified DNA can also be seen on an agarose gel with an ethidium bromide stain.

For primers F4/R2 and B4/B5 amplification reaction mixture was prepared in a volume of 25 µl containing 1X PCR buffer, 1.5 mM MgCl2, 200 µM each deoxynucleoside triphosphate, 20 pmol each primer, 1 U of Taq DNA polymerase and 3 µl of template DNA (~100ng). The following PCR conditions were used (Table 2).

4b). This clone was named C1and was taken for further characterization. Sequence analysis of C1 showed high homology (94%) with the VHH sequence published in the NCBI database.

A special concern should be provided for the selection and designing of the typing primer pairs to prevent variant primers interference during the PCR amplification stages. For each RV-strain, specific primer pairs involved, while, the annealing temperature of all primer pairs is similar to have a fruitfully multiplex-PCR process. Further innovative primers mix is hopeful for upcoming orphan strains identification and precise genotyping [14].