A Report On The Rna Essay

magine, 20 years from now, sitting in a cold doctor's office deciding the genes of your unborn baby, what color hair, eyes, speed of metabolism, height would you even know what to pick? Impossible you might say but in this day and age technology is growing ever so rapidly that picking the genetic makeup of your baby is closer than you might think. The technology is called CRISPR. This technology doesn't only have the ability to change physical traits, but genetic traits specifically genetic abnormalities and diseases. 20 years ago, no one would have ever thought we would have the answer to, in theory, cure every genetic disease from sickle cell anemia to cystic fibrosis. However, with great scientific breakthroughs comes questioning and

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

DNA is a term that has been used in science as well as in many parts of daily

This process uses enzymes to cut and insert piece of DNA into a plasmid vector. This vector is then transfected into cells. The different pieces of DNA that were being inserted into this plasmid were a control and three different mutants (an alpha-5-tailless mutant, full-length alpha-5, and full-length alpha-2). These pieces of DNA code for integrins. Once these pieces of DNA were inserted into the plasmid vector, the plasmid needed to be inserted into a cell where it would be able to replicate. In these tests, rat intestinal epithelial wild-type cells (cells that would be found in nature, not cells grown in the laboratory) were used. To insert the plasmid into these cells lipofectamine plus was used. Lipofectamine plus acts like a detergent and opens the membrane of the cell so the plasmid can enter. Also, the plasmids that were transfected into cells were all treated with G-418, an antibiotic. When the cells were plated and allowed to multiply, only those containing the plasmid with the antibiotic would live. This made it possible to know which cells actually received the plasmid and properly underwent mitosis, and to eliminate those cells that never took up the plasmid in the first place.

The detergent was used to isolate the cells we needed and chemically coated the cells to assist in the breaking down.

Experiment 1 & 2: The DNA concentration was 60 ng/µL (6.00*10^4 ng/mL). The total yield was 0.6 ng (60 ng/µL DNA /100 µL H2O). The DNA sample was divided by 100 µL instead of 25 µL of H2O because the sample was diluted 4 times.

Isolation of plasmid DNA from three cultures of E.coli using a method known as the alkaline lysis method.

- 350 μl of Buffer N3 (neutralization buffer) was added and mixed by inverting the tube for 6 times. Then white fluffy precipitates appeared that mean the plasmid DNA is unaffected.

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

We then added a wash solution to further purify the sample and centrifuged the solution again removing the eluate once more. We then centrifuged the tube for an additional 2 minutes and emptied the eluate. We then transferred the spin tube to a clean PCR tube. Which we then added 30 ul elution solution and incubated for 1 minute at room temperature and then handed off the sample to our TA to be centrifuged at a higher setting for 1 minute. We then discarded the spin column and prepared another PCR tube with 6 uL sterile water, 2uL DNA primer, and 2 uL of clean PCR product. We then handed off the tube to the TA to be processed by the sequencing

After the completion of electrophoresis, the gel is placed in denaturing buffer for half an hour and then placed into neutralizing buffer. After the gel is neutralized, it is placed onto a sponge wick sitting in salt solution. A nylon membrane is then placed over the gel. A stack of paper towels is then placed over the membrane. Sometimes a layer of glass is placed over the towels and weights are stacked onto the glass. This would ensure that the gel and membrane has even contact, but to much weight would result in crushing the gel. Once this is set up, the transfer is left overnight. During this time, capillary action with pull the salt solution in the dish below up through the gel. This process transfers DNA fragments from the gel onto the membrane filter. Any weights and the paper towels are removed the next morning. The nylon membrane is then removed and then rinsed with neutralizing buffer. The membrane is then placed on a UV transilluminator for cross linking. Once the membrane is removed from the transilluminator, the DNA is ready for hybridization.

This solution prevents endonucleases and proteins from being released during the lysis. With out this solution the quality of the DNA would be adversely affected. The time had to be carefully watched because prolonged exposure to the protease would cause some of the plasmid DNA to be nicked. 350µl of Neutralization Solution was added after five minutes to prevent nicking of the plasmid DNA. The bacterial lysate was then centrifuged at a maximum speed of 14,000-x g for ten minutes. About 850µl of cleared lysate is transferred into a prepared spin column, a spin column inserted into a 2ml collection tube. Only the supernatant was transferred into the column, the white precipitate was discarded. The supernatant was then centrifuged for one minute, and then put into a new collection tube with a 750µl diluted Column Wash Solution. The supernatant was centrifuged again for one minute; this wash procedure was then repeated with 250µl of the Column Wash Solution and centrifuged for two minutes. The spin column was put into a new, sterile 1.5ml microcentrifuge tube. The plasmid DNA was carefully eluted by adding

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double