Cloning Process : Buccal Swabs Or Whole Blood Samples ( 2-5ml )

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector . The vector is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation, optical injection and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to

By subjecting these bacteria through sudden temperature changes or heat shock, a difference in pressure in the outside and the inside of the cell is made, that causes pores, through which the mobile supercoiled plasmid enters. After normalizing the cell temperature, its walls will heal. When the E. coli has now taken up the plasmid with GFP, it will be able to grow in agar plates with ampicillin, an

Next heat shocks the tubes for 50 seconds, followed by icing for 10 more minutes. The heat shock increases the permeability of the cell membrane to DNA. Then add 250 milliliters of LB and incubate for 20 minutes. The 20 minute incubation following the addition of LB broth allows the cells to grow and express the ampicillin resistance protein, beta-lactamase, so that the transformed cells survive the subsequent ampicillin selection plates. Plate 100 microliters the + tubes evenly on two plates; 1 of LB and Amp, and one of LB, AMP, and ARA. Plate 100 microliters of the – tubes evenly on two plates; 1 of LB and AMP, and one on LB only.

How do you insert the plasmid inside the bacteria? What process do you use? (1/2 pt)

Firstly, the lab was done within a period of 75 minutes. Time efficiency was a factor that could have been more accurate when creating competent cells. Competent cells are able to take in extra foreign DNA. The procedure involved timing for series of events. Events such as: both tubes on the ice for 10 minutes, both tubes in the bath for 50 seconds, both tubes on ice for 2 minutes and both tubes at room temperature for 10 minutes.

This process consists of three major steps which are denaturing, annealing, and elongation. The cells are lysed before the denaturation step in order to obtain chromosomal DNA. In order to denature the DNA, or separate the two strands, the DNA is heated to 95 degrees Celsius. Once the denaturing is complete, the PCR tube is cooled to approximately 55 degrees Celsius during the transition to the annealing step. During this step, there are two primers: 27F and 1492R, which bind to the 16S gene on the strands of DNA. A high molar ratio of primers is used to ensure that there is attachment between the selected region of DNA and the primers as well as to ensure that the primers will hybridize. After the annealing step, elongation happens at around 75 degrees Celsius. A Master Mix is added during this step, which contains Taq polymerase and dNTPs. In order to replicate the sequence, Taq polymerase uses the dNTPs. Since the primer is on the 5’ end of the strand, the elongation is happening in the direction of the 3’ strand and the elongation is indefinite and these

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 .

The cells usually are put into a special liquid and sent to a laboratory for testing:

Then added 200 ul of P1 Buffer (Red) to the tube and re-suspend pellet completely. We then centrifuged at 11 rpm for 1 min and discarded the supernatant. Then we added 200 ul of P2 Buffer (Green) and mix by inverting the tube 2 - 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. (Proceed to the next step within 1-2 minutes) We then centrifuged at 11 rpm for 1 min and discarded the supernatant. We added 400 ul of P3 Buffer (Yellow) and mix gently but thoroughly. The sample will turn yellow when the neutralization is complete then incubate the lysate at room temperature for 1-2 minutes. We then centrifuged at 11 rpm for 2 min and transferred the supernatant to a Zymo-Spin column. Once transferred we spun for 30 seconds at 11,00 rpm and then spun with 200 ul of Endo-wash for 30 seconds then 400 ul of plasmid wash buffer for a minute. After that we spun our column empty for an additional minute to remove an excess alcohol. We then transferred out column to a clean centrifuge tube and added 30 ul of Elution Buffer to the column and incubated at room temperature for a minute and centrifuged for 30 seconds. The result was our plasmid DNA.

Reverse transcriptase polymerase chain reaction, also known as RT-PCR, has been recognized as a reliable, accurate, and sensitive method for quantifying gene transcription. Polymerase chain reaction, also known as PCR, is considered an essential tool in molecular biology that allows for the amplification of nucleic acid sequences. Specifically, the three main consecutively repeating steps in PCR are denaturation, annealing, and elongation. If the reaction runs with 100% efficiency, there will be a two-fold increase in target amplicons after each cycle of PCR. Therefore, with n cycles of the reaction, the copy number of the target sequences will be 2n. However, one of the main disadvantages of conventional PCR, also called end-point PCR, is that the results of amplification can only be visualized after all the cycles of amplification are complete (Nestorov 326).

The restriction enzyme is chosen based on the DNA sequence of interest. The type of buffer used in a restriction digest depends on the restriction enzyme being used. It is important to keep all components for the restriction digest in freezing temperatures until it is time to perform the experiment. The restriction enzyme, plus glycerol to keep the enzyme from freezing, is stored at freezing temperatures so that denaturing does not take place. Place all of the components in a tube and use a pipette to ensure that the components are mixed together. Next, the tube is incubated for an hour at 37o C, or the temperature specified for the restriction enzyme(s). In order to determine where the enzyme(s) cut on the plasmid and how many times the enzyme(s) made a cut, gel electrophoresis is employed.

The PCR cycle continues as DNA is copied exponentially. The original PCR methods allowed detection of DNA growth at the end of the process; however the introduction Real Time-PCR (RT-PCR) has allowed DNA amplification progress to be monitored throughout the assay (6).

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

Nucleic acid hybridization is a general method in determining the sequence of homologous DNA, it is also used to differentiate order of genes in a haploid set of chromosomes of a particular organism and the size of limitation fragments that contain such sequence. Meaning, it is possible to study the genetic differences between different organisms and individual here on Earth. A nucleic Acid Hybridization analysis uses five laboratory skills the Restriction fragment preparation: where a restriction enzyme is added to a DNA sample, Electrophoresis: separate the restriction fragment from DNA sample, Blotting: single strands are transferred onto special paper or nylon membranes through capillary action, Radioactive

Next step involve DNA sequencing of the cambial-region cDNA inserts. It was performed using PCR products as templates from the 59 end. Microtiter plates were loaded onto a robotic worktable. This worktable is the place where the PCRs, quality control and sequencing reactions performed automatically. How do PCRs performed? By using general vector primers and standard PCR controls