Genomic Dna Transformation Lab Report

After it was expelled back into the cup, 1 ml of the saline rise was transferred into a micro test tube in order for it to be spun in a balanced centrifuge. The micro test tube that contained the 1 ml of saline rise was inside of the centrifuge for 2 minutes, resulting in cells at the bottom of the tube. Since all of the cells were present at the bottom of the tube, the saline was poured off and the tube was vortexed to be sure that there were no clumps of cells. Afterward, the InstaGene Master Mix (which removes cofactor to inhibit DNA cutting enzymes) was vortexed with the saline rise in order to fully mix the contents of the tube (Bio-Rad Laboratories, Chromosome 16: PV92 PCR). The tube was then incubated at 56 degrees Celsius for 10 minutes to inactivate DNAses and put on a second heat block at 100 degrees Celsius for 5 minutes to disrupt cell membranes. The tubes were put into a centrifuge then cooled down in a 4 degrees’ Celsius fridge.

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In 1928, Fred Griffith first discovered genetic transformation by infecting mice with unencapsulated and non-pathogenic pneumococci (Lacks 2003). This was the start that opened up the field of biotechnology. Genetic transformation is a process where foreign DNA crosses a membrane of another cell and then alters the genetic material (Encyclopædia Britannica 2015). Genetic transformation can occur in a few different ways: projectile bombardment, electroporation, and heat shock (Weedman 2015). Heat shock is defined by increasing the temperature of cells environment making the plasma membrane become more permeability allowing new DNA to transfer into the cell (Weedman 2015). The cell receiving the new DNA, also known as competent cells, can either amplify the DNA or clone the DNA (JoVE 2015). The most common type of DNA used to perform genetic transformations is plasmids: small, round DNA molecules that still contains two strands of DNA that has the ability

Exploring the Expression of an Auxin Induced Gene pCNT103, a Constitutive Gene GapC and a Cytokinin Induced Cig1 Gene in the DNA and RNA Extracts of Differentiated Shoot, Root, and Callus Tissue of Nicotiana tabacum

A highly conserved gene will be used to identify a prokaryotic species isolated from the body. Fundamental lab techniques will be also explored and utilized, such as amplifying using PCR, cloning, and transforming the gene into a host cell. DNA electrophoresis and specific substrate plating will serve as analysis check points. The final product will be sequenced and compared to similar species to observe phylogenetic relationships.

For this experiment, whole bovine blood was used. The first process was to separate the blood into cellular and plasma fractions. 100 µL of whole bovine blood was transferred into a yellow microcentrifuge tube that was labeled WB using a P-200. 50 µL of whole blood was added to a blue microcentrifuge tube labeled WB. Both tubes were capped and placed in ice. 2 mL of the remaining blood was transferred into a Clear 2 mL tube using a P-2000 and centrifuged for 5 minutes at 8000 RPM. Afterward, 800 µL of the supernatant from the Clear tube was transferred into a yellow tube labeled WP and 50 µL of the supernatant was added to a blue tube labeled WP. These tubes were then capped and kept on ice.

Purpose The main purpose of this experiment was to test and observe how DNA molecules are being tested or separated. Introduction The final goal of this lab was to successfully measure the size of different samples of DNA placing each samples into a well in agarose gel and running a current through a charged chamber.

To understand the transformation lab we did, you need some background information to help understand what we did. The DNA structure is formed in a double helix which means it has two strands and consists of nucleotides. Each nucleotide contains deoxyribose sugars that are bonded by phosphodiester which bond to a phosphate group and a nitrogen base. The nitrogen base matches up to the nitrogen base on the opposite strand of the double helix. There are two types of nitrogen bases that occur, purine which is either A or G which form a hydrogen bond with pyrimidine which is either T or C. When DNA is replicated the hydrogen bonds that hold the strands together break down by an enzyme and then the RNA primase is added so DNA polymerase 3 can attach

After 30 seconds we expelled the solution back into the cup and swirled gently to mix cells. Second, we transferred 1000µL of the solution into our labeled microcentrifuge tube. Third, centrifuge for 90 seconds at full speed then pour off most of the supernatant into the paper cup without disturbing the pellet. Fourth, we have to resuspend the cells then withdraw 30µL of the cell suspension and add it to the PCR tube with 100µL Chelex. After we put the labeled tubes in the thermal cycler which was set to 99ºC for 10 minutes, once done shake vigorously. Fifth, we centrifuged the sample for 90 seconds at full speed once done we transferred 30µL of the clear liquid into a new 1.5mL tube and labeled it with our assigned number. Store on ice until ready for it in part

This two-week lab had an overall theme of DNA analysis. Humans can distinguish between five different tastes: sour, sweet, salty, bitter and umami. In the 1930’s, a man named Arthur Fox created a molecule that some people can taste and others cannot, it’s called phenylthiocarbamide or PTC. It is based off a gene variation, whether or not someone can taste it. Tasting PTC is dominant and not tasting it is a recessive gene. The TAS2R38 gene can also be shown as incomplete dominance which means that the person can barely taste it. This can be tested by collecting a sample of cells from the human mouth and extracting the DNA by boiling it with a Chelex resin. This binds to metal ions to help

This was filtered through a cheesecloth and the supernatant was collected into a 50mL centrifuge tube. This was centrifuged for 10 minutes at 6,000rpm. The supernatant was discarded, and the pellet was broken up and diluted to 50mL with cold buffer solution and centrifuged for 10 more minutes. Once again the supernatant was discarded and the pellet was broken up and diluted to 50mL with 2.6M NaCl solution. This was centrifuged for 15 minutes, and the supernatant was saves without the pellet. The supernatant was kept in a beaker on ice and 100mL of ethyl alcohol was added. The DNA was precipitated and extracted with a glass rod. This DNA was added to a tube with 10mL distilled H2O. Using seven tubes, 2mL distilled H2O was added to tubes #2-#5 and 2mL standard stock solution was added to tubes #1 and #2. The 2mL from tube #2 was added to tube #3, and 2mL of this was added to tube #4 and mixed. Then, 2mL of DNA was added to tubes #6 and #7. We discarded 2mL from tube #7 and mixed the tube. We added 4mL of Dische reagent to all seven tubes and heated them for 10 minutes. Once they were cooled, the absorbances were recorded at 500nm, using tube #5 as a blank. Tubes #1-#4 were plotted to determine the concentration of DNA from tubes #6-#7, and the concentration of DNA per gram of deer spleen tissue was

For this part of the lab we used well-fed tetrhymena and 5% concentration of India ink throughout the experiment. The first two microcentrifuge tubes were positive and negative controls. The negative control contained only 0.2mL of the well-fed tetrahymena. The positive control contained 20 microliter of well-fed tetrahymena and 20 microliters of PFA in the microcentrifuge tube. We added 0.2mL of tetrahymena to the remaining centrifuge tubes. The third microcentrifuge tube (first condition) received 10 microliters of glucose. Then the fourth microcentrifuge tube received 10 microliter of colchicine (second condition). The fifth centrifuge was placed in an ice bath (so that the tetrhymena could be in a cold environment, also is the third condition).

Each 1.5 ml microcentrifude tube containing 50 µg myceliums, 50 µl of Lysozyme, 450 µl of Lysis solution and 3 µl of RNase A was kept for hour at 37 0C on water bath, vortex several times in duration. Then, addition 250 µl of 2% SDS, gently invert before addtion 250 µl of mixing Phenol- Chloroform- Isoamylclcohol ratio is 25:24:1, vortex and centrifuge at 12,000 rpm in 10 minutes then transfer supernatant on up layer to a new 1.5 ml microcentrifuge and repeat that step again before the next step to completely remove unexpected components. Subsequently, 0.1X of 3M Sodium acetate pH 4.8 was supplemented,

Once the pellet had formed I carefully poured out the liquid that was above it leaving just a little bit of liquid in the tube with the pallet. Then taking a 200 microliter PCR tube, using a micropipette with a fresh tip, I first added 100 microliters of Chelex solution, which is used for DNA extraction when preparing for PCR, and then 30 microliters of my cell suspension which was in the microcentrifuge tube. That PCR tube was placed in the thermal cycler for 10 minutes at 99 degrees celsius to break down the cell so DNA can be released. Once the 10 minutes was up the tube was shaken up in the vortex for 5 seconds. The tube was then put in the microcentrifuge for 90 seconds to form another pellet. Once the 90 seconds was up I took the micropipette again with a new tip and transferred 30 microliters of the liquid on top of the

Transformation of the pAMP to the E. coli showed growth in all plates that did not contain ampicillin, which was to be expected as E. coli with or without the ampicillin resistant DNA would be expected to survive in an environment that did not contain the ampicillin. Additionally, E. coli that was not transformed did not survive in plates containing ampicillin; a fact that we had already predicted as the bacteria has no genes protecting it from the deadly antibiotic. However, we found slightly different results when the bacterium that was transformed with the pAMP was cultured in plates (labeled A and B) containing ampicillin. While we did see growth on the B plate, we did not see any growth on plate A. To explain this, we also must take into account the fact that different concentrations of bacteria were used to culture each plate. Plate B was cultured with 100 µL of bacteria while plate A only contained 10 µL of bacteria and 90 additional µL of luria broth. The bacteria without the pAMP transformation acted as a control when we tested the