Amplified Using Phusion High-Fidelity DNA Polymerase

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 2nd method consisted of purification of the plasmid DNA provided (bacterial culture). 1.5 ml of 3 bacterial culture was added to 3 different centrifuge tubes and then centrifuged for 1 minute at about 8000 xg. The supernatant material from all 3 tubes was then discarded into 3% Virkon solution and the tubes were placed back in the rack. Then 250 µl of P1 Lysis Buffer was added to the tubes and vortexed followed by an addition of the same amount of Lysis Buffer P2 and mixed gently by inverting the tubes 6-7 times, the tubes were then left to incubate for 5 minutes at room temperature. 300 µl of Neutralisation Buffer P3 was added and then mixed thoroughly by inverting the tubes. All the tubes were now centrifuged at 11,000 xg for 5 minutes taking into consideration that the centrifuge machine was balanced when used to avoid any incomplete mixture. The tubes were then removed and 750 µl was carefully added to 1.5 ml spin columns as to not disturb the white residue (Na), the tubes were then again subject to centrifuge for 1 minute at 11,000 xg and the flow through discarded after the run. 500 µl of Wash Buffer PW1 was added and centrifuged for a minute (at the same xg), after

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.

For the quantitation of DNA both PCR and plasmid samples (1μL) were placed on the Nanodrop spectrophotometer separately and the absorbance was set to 260 nm which was measured for A260/A280 ratio. This was followed by the digestion of restriction enzyme. For this, both plasmid (1.6μL) and PCR (0.3μL) samples were mixed with 10x NE buffer, EcoRI and water separately in an eppendorf tube which was quick spin for 30 s at 1000g followed by incubation in Forma scientific water jacketed incubator for 15 min at 37°C. Before the

Plasmids were extracted using the three-step alkaline lysis procedure as outlined by Kado and Liu, 1999. The extracted plasmids were then digested using 1 U of each XhoI and XbaI, respectively according to manufacturer’s (New England Biolabs) recommendation and ran on a 1% Tris Acetate EDTA (TAE) agarose gel. 1 μl of BioLine Hyperladder I was used as the DNA marker.

The small amount of colonies were put into a labeled PCR tube which contained: 10 µl GoTaq Master Mix, 1 µl F primer for the SSU, 1 µl R primer for the SSU, and 7µl of H20. The tubes went through a various thermal cycler program. 10min at 95 celsius, 30 seconds at 95 celsius, 30 seconds at 55 celsius, 2 minutes at 72 celsius, and 10 minutes at 73 celsius. The tubes were placed in a refrigerator until gel electrophoresis.

PCR was done in a final volume of 25 µL containing 5 µL of bisulfate modified DNA, 1 µL of each primer (1 µM), 12.5 µL of 2X Super Hot PCR Master Mix (Bioron, Germany) and 5.5 µL of H2O. PCR protocol was performed at 95 °C for 10 minutes, then 40 cycles at 95◦C for 30 seconds, annealing temperature for 30 seconds at 61°C for methylation and nonmethylation specific primers of MGMT and at 58°C for methylation-specific primers of ERCC1 or at 54°C for nonmethylation-specific primers of ERCC1, 72◦C for 30 seconds, and final

In order for DNA samples from suspect one and suspect two to be digested by two different restriction enzymes, four reaction tubes were required, labeled 1-4. In each reaction tube, with a micropipette, ten µL of reaction buffer was used. All the samples were prepared based on the given chart (shown below as Table 1) (Upadhyaya, 2017, p. 58). As far as all four having the same enzymes that was the end, so to not cross-contaminate, the micropipettes tips had to be changed each time (Upadhyaya, 2017). The reaction tubes one and three contained 15µL of enzyme 1 and enzyme 2 was added to reaction tubes two and four. Then, reaction tubes one and two were filled with 15µL of

The article describes using the method of gel electrophoresis to separate DNA fragments to identify and analyze certain sequences. The DNA fragments were broken down using a restriction enzyme from Haemophilus influlenzae, allowing the researchers to see that these restriction enzymes can be used to study DNA and its structure. The first step of the gel electrophoresis was to set the gel matrix. Agarose was used to separate DNA molecules, and acrylamide was used to separate proteins. The gel starts off as a liquid, which was poured into a molding tray. A comb was placed in the liquid matrix so that when the matrix solidified, wells formed to load samples in them. After the gel solidified, the mold was removed and placed

CsA concentration varied between rounds (Table 1). Library was ordered from Integrated DNA Technologies (IDT). N30 library (30 unknown nucleotide bases): 5 '-GGA GGC TCT CGG GAC GAC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GTC GTC CCG ATG CTG CAA TCG TAA-3 '. Capture Sequence: GTC GTC CCG AGA GCC ATA/3BioTEG/. Primer_P1: GGAGGCTCTCGGGACGAC. Primer_P2: TTACGATTGCAGCATCGGGACG. P2_biotin: /5BiosG/TTACGATTGCAGCATCGGGACG. All oligo’s (primers, library, and capture-strand) were ordered from IDT . Bio-capture used: 5’ biotin from IDT. Two SELEX buffers were used: SELEX 1 buffer (10 mM HEPES, 150mM NaCl, 5mM KCl, and 2mM MgCl2) and SELEX 2 buffer (10mM HEPES, 150mM NaCl, 5mM KCl). The composition of the PCR mix was as follows: 10% 10X buffer, .5% Taq, 1% P1, 1% P2, 2% dTNP, and 80.5% water. All gels were run in .5X TBE buffer and 3% agarose gel. Columns used: Micro Bio-Spin™ Chromatography Columns. PCR polymerase used was Choice Taq from Denville Scientific. DNTP is also from Denville Scientific. Streptavidin Beads are from Sigma Aldrich.

To determine if our DNA sequences were properly extracted and amplified, we used gel electrophoresis. A 1% agarose gel was made using 1 gram of agarose powder per 100 ml of 1x TBE buffer. The solidified gel was placed in the electrophoresis tank containing 1x TBE buffer. Our samples were prepared by mixing gel loading solution with our PCR products (3 µL gel-loading solution, 5 µL PCR product). The samples were loaded in a selected order (table 2) and the gel was run between 30 minutes to an hour at 70-80 V (Lab 8, 2017)

In experiment of Bacterial plasmid DNA extraction, solution 1 which consists of ice-cold resuspension buffer helped corrupting the cell membrane. Because cell membrane has not been broken down yet the collected blend is clear. However, later on inclusion of Solution 2 which is lysis buffer the cell membrane was broken. The solution 3 that is a neutralization buffer was added and it provided the contaminated genomic DNA to precipitate. Using centrifugation method provided the plasmid DNA to isolate from cell debris and genomic DNA. Protein contamination was tried to prevent using ethanol.

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

Gel purification allows us to isolate and purify DNA fragments based on size. The DNA bands from the agarose gel are cut out and purified using some procedure. The band was extracted by using the QIAgen gel extraction protocol. The DNA bands after purification for CD01 and CD02 were shown in Figure 4.3. This steps also considered to be vital components in molecular biology technique as it can help determine the success or failure of the downstream

Agarose gel electrophoresis used for the separation of the fragments was based on the migration of the negatively charged DNA to the positively charged anode. DNA has a uniform mass/charge ratio therefore allowing the molecules to be separated by size within an agarose gel such that the distance travelled is inversely proportional to its molecular weight. The rate of migration is controlled by the following; size of DNA molecule, agarose concentration, DNA conformation, voltage applied presence of ethidium bromide, type of agarose and electrophoresis buffer.