Biochemical Techniques for the Extraction of Escherichia Coli Genomic DNA

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.

Introduction: All organisms have DNA (Deoxyribonucleic Acid) in the nuclei of their cells. The aim of this experiment is to extract this DNA using common household detergent to breakdown the cellular membrane then through the use of ethanol to extract the DNA from the Kiwi and Strawberry fruit. After the DNA has been extracted the DNA will be visualised by agarose gel electrophoresis which is done by adding a dye to the extracted DNA then being placed in the gel, once the negatively charged DNA has been placed in the gel it will slowly move towards the positively charged end (anode) then is viewed under an ultraviolet light then compared to the control of known lengths of DNA to show the size of the unknown DNA samples.

There are three specific steps required to isolate DNA from its cellular contents. The steps used to remove and expose DNA from its cell are: breaking down the food type you are using by crushing it, for example a banana or strawberries, exposing the substance to a sodium chloride (NaCl) solution, subjecting the product to detergent solution (dH2O), filtering the solution and lastly, the addition of ethanol. When beginning with a solid substance, such as a banana, crushing the substance allows for

(PCR), which isolates small fragments of DNA that have a high degree of variability from

In order to extract DNA from any living thing, we needed to first gather the materials. Then we began the experiment. Step 1, put in a blender 1/2 cup of split peas (100ml), 1/8 teaspoon table salt (less than 1ml), and 1 cup cold water (200ml). Next, we blended the materials on high for 15 seconds. This allowed the pea cells to separate from each other, so we now had a really thin pea-cell soup. Step 2, poured our thin pea-cell soup through a strainer into another container. Added 2 tablespoons of liquid detergent (about 30ml) and swirled to mix. We then let the mixture incubate for 7 minutes. Poured the mixture into test tubes containers, and filled each about 1/3 full. Step 3, added a pinch of meat tenderizer to each test tube and stirred gently to make sure we didn’t break up the DNA. Step 4, tilted our test tube and slowly poured rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Poured until we had about the same amount of alcohol in the tube as pea mixture. Alcohol is less dense than water, so it floats on top. From there we looked for clumps of white stringy stuff where the water and alcohol layers meet. The white stringy stuff was tangled DNA molecules. Thus, we completed DNA extraction. As we performed the experiment we made no changes to the original protocol.

coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.

Next the tubes were placed in an ice bath, while obtaining a sterile loop to swipe a single colony of E.coli to put into the tube. After gently swiping a colony onto the loop, it was then spun in the +pGLO tube to get it to come off, returned to the ice bath. Next using a different sterile loop, it was swooped it in a container labeled pGLO plasmid DNA and again spun it ONLY into the tube with the solution labeled +pGLO to get it to come off. After about 10 minutes on ice the tubes were then placed into a 42ºC water bath for 50 seconds exactly, and immediately after placed them back into the ice bath. Finally, after 2 more minutes in the ice bath the tubes were separated into 4 containers. 250 ul of +pGLO solution was added to the containers containing +pGLO, LB broth, with ampicillin and +pGLO, LB broth, ampicillin, with arabinose. Also 250 ul of the –pGLO solution was added to the 2 containers containing LB broth, and ampicillin, with LB broth. Using a sterile loop for each plate the solutions were spread out gently and thoroughly on to the containers with agar. After the containers were incubated in 37ºC for at least 24 hours, the results were observed and disposed of (Weedman,

The DNA extraction results, along with the PCR product, did not fare well. There was not enough product produced to be viable in the later stages of the experiment, so a backup was used in place of the original product.

This experiment’s purpose is to identify and separate DNA macromolecules with a homemade gel and electrophoresis chamber. After separating DNA with a homemade gel and electrophoresis chamber, it must be compared to the regular gel electrophoresis chamber. In order to create a homemade gel electrophoresis chamber, five 9-volt batteries were connected and attached to stainless steel wire which were submerged in the 20x buffer as well as the gel. The two wires acted as the positive and negative electrodes as seen in a gel electrophoresis chamber. After the experiment was done, the homemade gel electrophoresis chamber was proven to mock the regularly used electrophoresis chamber. The experiment suggested that the results would not change if the

Initial trials were performed in class of DNA extraction. This time strawberry was used. The process that was undertaken

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

Deoxyribonucleic acid is a very crucial key for life within organisms. DNA is a complexed, long-chained molecule that encodes the genetic characteristics of a living organism.1 DNA contains ribonucleic acid and proteins within chromosomes that are found within the cell’s nucleus. In typical humans’ cells, we contain 23 pairs of chromosomes, for a total of 46 in which we get half from our mother and the other half from our father. DNA acid is a polymer made of four nucleotides: Cytosine, Thymine, Adenine and Guanine. DNA is usually double-stranded, with A and T being hydrogen bonded to each other, and C and G being hydrogen bonded. In this experiment we observed DNA composition by High-Performance liquid chromatography (HPLC). HPLC is a quantitative analysis technique that is commonly used to separate and identify individual components within a mixture. High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).2 With the use of HPLC we were able to observe different amounts of nucleotides that were present in calf thymus DNA.

Besides, Solid-phase extraction methods for DNA have been developed in recent years in formats that enable high-throughput DNA extractions. Moreover, Solid phase extraction (SPE) is widely used for clean-up in food analysis, and has largely replaced classical gravity column chromatography. 6

Results showed that approximately twice as much nucleic acid was extracted from the direct transfer method (1523 ± 225.9 ng, A260/280 = 2.03 ± 0.02) compared to in-channel lysis (647 ± 5.7 ng, A260/280 = 1.99 ± 0.08) with statistically significant differences (n = 5, p-value < 0.05). For the normalization of data in Figure 3.10(c) the number of cells equivalent to the initial cell seeding density was pipetted directly from cell suspension into lysis buffer. The data are presented as the extraction efficiency with respect to the control. The extraction efficiency of direct transfer and in-channel method was 80% and 35%, respectively. The purity of the extracted nucleic acid between the methods was comparable, but the direct transfer method was simple to implement and did not require sample handling or fluid manipulation. The straight channel used here represented a conservative extraction because more complicated branched networks can lead to sample losses from dead volumes, external tubing, and large surface area-to-volume ratios microfluidic channels. The open cell access enabled by SEAM simplified the experimental workflow for cell processing required for