Cdna Synthesis Lab Report

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).

Part 3 of this experiment calls to isolate the DNA from C. Elegans. To do so, we start by labeling three PCR tubes with the group number. One of the tubes is labeled “W” for wild type, one is labeled “R” for

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

Also (5μl) of DNA template that extracted from stool samles was added then 1.5 μl of each type of Primers(forward and reverse)added to the master mix and then blend well using Exispin vortex centrifuge ,then this tubes would transferred to the Thermocycler machine, which has been programmed by the previous program for amplified of ITS1 region.The PCR products were electrophoresed in agarose gel and visualized on UV trans illuminator and then photographed using photo documentation .

NaOH is then applied for cell lysis and the ‘unzipping’ of dsDNA to ssDNA. The ssDNA may then be used to isolate and replicate the PCR product through the use of PCR and site specific primers, using 2 specific primers to isolate both sides and ends of the mtDNA D.loop, multiple runs of PCR are taken to receive multiple copies of the PCR product. The following sequence primers are used to isolate the PCR

Extracted RNA (1 μg) was reverse transcribed using high-capacity cDNA reverse transcription kit, supplied by Applied Biosystems, USA according to the manufacturer's instructions. The cDNA product was diluted in 200 μL Nuclease-free H2O.

Total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, CA). First-Strand Synthesis System for RT-PCR (Invitrogen) was used to synthesize cDNA from 1.5 μg total RNA according to the oligo (dT) version of the protocol. Real-time PCR was performed using CFX Fast real-time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA). The following cycle parameters were used for all experiments: 20s at 94°C, 30s at 60°C, and 30s at 72°C for a total of 45 cycles. The relative level of mRNA for a specific gene was normalized to GAPDH levels. Table 1 shows the sequences for all primer sets used in these

A fragment of DNA was then amplified using Polymerase Chain Reaction (PCR) with a forward primer and a reverse primer. PCR is a technique used to make as many copies as possible of a specific section of the DNA extracted. The five necessities for PCR to be successful is the DNA template that one wants to copy, a buffer, nucleotides (ATCG), polymerase, and primers. PCR occurs in 3 stages: (1) Denaturing, (2) Annealing, and (3) Extending. During the Denaturing stage, the stand containing the DNA template (in this case the fetus’) is heated to separate the double stranded DNA. Next during the Annealing stage, the stand is then cooled to allow the primers to attach to a specific location on the single stranded DNA template (primers are usually 20 to 30 bases long). Lastly during the Extending stage, the stand’s temperature is increased to permit the making of new DNA by a specific DNA polymerase. The result is multiple new strands of DNA that has been successfully copied (What is PCR, WEB), followed by electrophoresis in 1% agarose gel. Electrophoresis is a technique commonly used to separate DNA, RNA, and proteins according to their sizes. The result was then purified with a PCR purification kit (removing any remaining DNTPs, or nucleotides that could result in contamination). The products were

In addition, one PCR condition will be varied to determine its affect on amplification, and which conditions are optimal for PCR amplification [1]. This particular part of the experiment brings into light the optimal conditions for PCR, as well as, strengthening understanding of amplification with PCR.

Agarose gel was prepared to use for the detection of geneomic DNA by adding 1gm of agarose to 100 ml of 1X TBE buffer and it is dissolved by heating at boiling temperature. Then the agarose was left to cool at 55C°, before pouring in a casting plate to solidify. A required comb was placed near one edge of the gel, and the gel was left to cast. 1XTBE was poured into the gel tank and the gel plate was placed horizontally in an electrophoresis tank. The DNA samples were prepared by adding 1µl of loading buffer and mixed with 5µl DNA samples, and then the samples were added carefully to individual wells. Power was turned on at 45V for 15minute and 85V for 1 hours to run DNA or at 5-8v/cm. Agarose gels were stained with ethidium bromide by immersing

350 of each sample are loaded on the gel. Based on Genomic Solutions, once fixing the changes of fresh 40% methanol and 10% glacial acetic acid for every 12 hour each, the gels were stained for 12 hours with Sypro Ruby solution in the dark. The gels were incubated in 10% methanol: 6% acetic acid for 4 hours for destaining procedure. The gels are the imaged by using ProPick Workstation. Triangulation and robotic excision are conducted for identification of protein spots. It was then submitted to tandem mass spectrometric analysis.

60 µM of Lysozyme (10 mg/ml) was added to 6 ml of the solution. 60 µM of 0.1 M Deoxyribonuclease I, 25 mg/ml (10,000 U/ml) was added after and incubated for two minutes. It was followed by addition of Ethylenediaminetetraacetic acid (EDTA) and incubated for 10 minutes. 60 µM of 1M Magnesium sulfate were added, carefully mixed and incubated for 20 minutes. The centrifugation technique was completed with Sorvall RC 5C Plus (code 3.19.5.06) with Rotar Sorvall SM-24, Autoclovable 121 C at 12,000g speed, at 4oC for twenty minutes. The Enzyme Stage 1 (0.3 ml) was extracted for additional analysis. The dialysis procedure was performed using Dialysis tubing Seamless-cellulose, width 25mm, diameter 15.9mm, 4.8 nm pore diameter, that retained materials with 12,000 or higher MW. The length of the tubing was cut and was approximately 14 centimeters, soaked in water, and after was filled with the retentate and left in a beaker covered with plastic wrap. The dialysate was the 10mM Tris-HCL, pH 7.4 and 10mM Magnesium sulfate. The buffer was replaced few times within the

All DNA tests were changed in accordance with 100 ng/μl. A 1000 ng (1 μl) aliquot of every example's DNA was utilized for a 50 μl PCR response. The 16S all inclusive eubac-terial preliminaries 530F (5'- GTG CCA GCM GCN GCG G) and 1100R (5'- GGG TTN CGN TCG TTG) were utilized for increasing the 600 bp area of 16S rRNA qualities. HotStar Taq Plus Master Mix Kit (QIAGEN Inc.) was utilized for PCR under the accompanying condi-tions: 94°C for 3 min took after by 32 cycles of 94°C for 30 sec; 60°C for 40 sec and 72°C for 1 min; and a last prolongation venture at 72°C for 5 min. A second-ary PCR was performed for FLX (Roche, Nutley, NJ) amplicon sequencing under the same condition by using designed special fusion primers with differ-ent tag sequences: LinkerA-Tags-530F and LinkerB-1100R. The resultant individual sample after parsing the tags into individual FASTA files was assembled using CAP3. The ace files generated by CAP3 were then processed to generate a secondary FASTA file containing the tentative consensus (TC) sequences of the assembly along with the number of reads in-tegrated into each consensus. The TC was required to have at least a 3-fold coverage. The resulting TC FASTA for each sample was then evaluated using BLASTn (Altschul et al., 1990) against a custom data-base derived from the RDP-II database (Cole et al., 2005) and GenBank website (http://www.ncbi.nlm. nih.gov/). The sequences contained within the

Hair, skin, eyes. Do you have any idea why we have the physical features we possess? Well, that all lies in our DNA. RNA and DNA, also known as ribonucleic acid and deoxyribonucleic acid, both work hand in hand within our body. RNA is what translates the genetic information into specific instructions for the assembly of proteins. DNA on the other hand is what contains all of our genetic information necessary to build cells, to combine them into an organism, and to maintain them. Even though DNA and RNA may seem very similar at first glance, there are many differences in various ways including its structure, purpose, and function.

Further, to measure the transcript levels of foxh1 in X. tropicalis tissue fragments (early gastrulae stage) and transcription levels of the members of PouV family, the quantitative RT-PCR was used. While this method is known as a sensitive and powerful tool for analyzing of RNA samples and it has a remarkable potential for quantitative applications, its technical features require a complete knowledge of the system. Experimental variations in individual RT and PCR efficiencies should be corrected in the quantitative RT-PCR. Moreover, the expression of one or multiple housekeeping genes is essential as a reference for the analysis of testing genes’ expression levels. One of the other strong points of this research is, in fact, having appropriate control genes in the study. For example in the associated results for Figure 1-A, the transcription level of the ribosomal protein L1 (rpl1) i.e. the control gene is clearly provided.