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Chapter 17 introduction to microbiology
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Recommended: Chapter 17 introduction to microbiology
Bacterial Genetics Aim: The purpose of the lab was to alter the plasmid DNA of bacterial cells and to observe any variations in the phenotype of the bacteria expressed in the plasmid after incorporating new genes. The lab consisted of three parts. In the first part, plasmid DNA was incorporated into bacterial cells. The second part consisted observing the new phenotypic traits on agarose plates, and isolating the plasmid DNA from the transformed bacterial cells to be used in PCR reactions. The final part was analyzing the PCR reactions on agarose DNA gel electrophoresis. Materials and methods: Part 1 - Bioluminescence Materials: • sharpie • 37 oC water bath • Ice • Sterile transfer pipette • Foam tube rack • Transformation solution …show more content…
Determine the concentration of DNA using a nanodrop apparatus. Dilute plasmid DNA to 20 µg/mL by following directions from the instructor. 2) Materials: Assembly of PCR reaction • Edvotek PCR pellet • Small PCR tube • Micropipettors and tips • Primers • Plasmid DNA from Week 6 • Microcentrifuge • Thermal cycler • 10X gel loading solution Procedure: Activity 2 1. Put on appropriate lab safety attire and gloves 2. Add Edvotek PCR pellet to 0.2 mL PCR tube 3. Add 20 µL of the primer pair. 4. Add 5 µL of plasmid DNA 5. Dissolve pellet by mixing. Put the tube in a microcentrifuge to spin and gather the remains at the bottom of tube. 6. Write initials on the tubes with sharpie. 7. Put the tubes on a thermal cycler and start it. Part 3- Agarose gel electrophoresis Materials: • Agarose gel electrophoresis apparatus • 1% agarose gel in 1X TAE buffer & SYBR® Green • Microipettors and tips • UV transilluminator • PCR samples with added tracking dye Procedure: 1. Put on appropriate lab safety attire and gloves 2. put 20µL of PCR reaction into a section in the agarose gel with a micropipettor. 3. Load DNA ladder in one of the agarose gel wells. 4. put agarose gel at 100 volts and run for an hour. 5. Observe agarose gel on UV transilluminator to examine results of the polymerase chain …show more content…
The first step was making the cell competent so it can take up DNA. Then plasmid DNA was introduced to the cell and the process of transformation was underway. We then viewed any phenotypic traits. We look for growth and bioluminescence in the cells. Both positive plates with amp displayed growth and one of them was bioluminescent because of the ara presence. The two negative plates had no bioluminescence and the plate with amp had no growth. The final part of the objective was also met because we examined the PCR reactions on the agarose gel electrophoresis. The whole experiment can be applied to professional life in the future because the lab gave me a glimpse of what it means to be a scientist. In the future, I plan on becoming a doctor and members of the healthcare environment are thought to be scientists. This lab was just a small piece of what is going to prepare to become what I want in the near
The pK184hlyA and pBluescript plasmids were cut using EcoR1 and Pst1 restriction enzymes that cut at specific restriction sites. Gel electrophoresis was carried out to check if the digestion procedure was done successfully.
After 48 hours of incubation the agar plates were viewed. Individual colonies were tested for successful isolation by gram staining and then viewing the stained bacteria under a microscope. Isolation was successful. One colony of each unknown bacteria was transferred to an agar slant for growth. The agar slants were stored at room temperature over the weekend so that they would not grow too much.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
...q DNA polymerase to each tube while disallowing the tubes to cool and without taking
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
5.) One at a time, place your test tubes in the water bath and heat the first test tube to 25 , the second to 50 , the third to 75, and the last to 100 degrees c. Remeber to stir with your stirring rod every so often.
6. Unscrew cap on Penicilium italicum culture tube with one hand and flame the mouth of the tube.
In a 100ml beaker place 50mls of water, measure the temperature of the water and record this initial temperature onto a table. Set the timer and add one teaspoon of Ammonium Nitrate to the water, stir this continuously until the Ammonium Nitrate has dissolved.
It is used in many labs and only requires the DNA in question, primers that anneal to the beginning and end of the target genes, Thermus aquaticus, Taq DNA, a heat stable DNA polymerase and all four of the deoxyribonucleate triphosphates. There are three steps in the PCR reaction denaturation, hybridization and DNA synthesis. During these steps the DNA is separated or denatured into two strands, hybridized, where the two single strands are complimentary paired to the respective primers, and then the DNA is synthesized with Taq DNA. This is considered one cycle, and it can commonly take 50 cycles to amplify enough DNA to be used. When the PCR is completed a gel electrophoresis is run. The PCR product is put in a specially formed agarose gel that will allow electricity to flow around the gel and DNA and force the DNA to travel down the gel resulting in white bands depending on their electronegativity. When the DNA is transformed from plasmid into the yeast we use salmon sperm to protect the nucleus from becoming degraded and the plasmid lost. This increases the efficiency of the DNA because the sperm DNA will adhere to the yeast cell wall and allow the plasmid to bind to the
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
Create wells: put a comb template in the middle of the tray; wait until the mixture becomes solid. After, remove the comb standing straight. 4. Remove rubber ends: transfer the gel tray into the horizontal electrophoresis and fill it with the concentrated electrophoresis buffer. 5. Materials and methods: Experiment: 1st, prepared milk samples should be already done by the teacher.
Step 1 is repeated by using different yeast strains, a pet 1 and M240 into all 6 conical
tube. Add 6 mL of 0.1M HCl to the first test tube, then 0.1M KMnO4 and