3.0 Material and Methods 3.1 LB Broth and LB Agar Preparation LB Broth powder and LB Agar powder (Sigma-Aldrich) were used for preparation of LB Broth and LB Agar respectively. For LB Broth, 35 g of LB Broth powder was added into 1 L of ddH2O. The mixture was heated up to boiling until all the LB Broth powder was dissolved. For LB Agar, 25 g of LB Agar powder was added into 1 L of ddH2O. The mixture was then allowed to stir to suspend. Both LB Broth and LB agar mixture were allowed for autoclave for 15 minutes at 121 °C to sterilize. Both solutions were allowed to cool slightly before making additions, such as antibiotics (ampicillin). LB agar was prepared by pouring LB agar solution into petri dishes and allowed to solidify. The petri dishes were seal with parafilm tape and inverted before store at 4 °C together with LB Broth. 3.2 Colony Screening from Glycerol Stock Petri dishes contain LB Agar that was prepared was used for streak plate method. Next, flame the inoculum loop and wait for it to cool down. The inoculum loop takes a drop of the liquid culture medium (Glycerol Stock) and spread carefully in a line across the surface of the agar. Sterilise the loop in the flame again and allow it to cool. Turn the petri dishes so the previous line can be the start of the next ones. After repeated several times, close the …show more content…
The mixtures were centrifuged at 13000 rpm for 2 minutes at room temperature. The upper aqueous solution was transferred to another sterile 1.5 ml microcentrifuged tube. Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes. Again, the aqueous solution was transferred to a new 1.5 ml microcentrifuged tube and 1/10 volume of 3M Sodium Acetate Solution was added. Then, 2.5 volumes of cold absoluted ethanol were added to precipitate the DNA. The mixture was incubated in -20 °C for overnight or -80 °C for 1-2
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
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
The gels were run at 90-100 volts for 1-1.5 hours. Upon completion of the experiment, we were able to examine the DNA. First, the electrophorese. revealed that three of the fourteen samples were homozygous while the other eleven were
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
coli cells were grown at 37 degrees Celsius with calcium ions. These cells were then incubated in ice and placed into 4 polypropylene tubes. Each tube consisted of 25 microliters of E.coli cells. Two of the four were labeled with (+) while the other two were labeled (-). Positive was with samples that contained 100 pg/ microliter of pGLO, while negative was for the samples that did not. The 4 tubes were incubated in ice for 30 minutes, then heat shocked for 30 seconds at 42 degrees Celsius. The 4 tubes were then returned to the ice bath for 5 minutes. After the removal, .975 microliters of LB media was added to each tube at room temperature. The tubes were then shook at 225 RPM at 37 degrees Celsius for one hour. 300 microliters of cells were then extracted from the tubes and then placed in agar plates. The cells were spread across the plate and the incubated overnight at 37 degrees
Sterile compounding is the preparation of products that should be free from all viable forms of life. There are more stringent requirements for sterile compounding than there are for non-sterile compounding. Staff must be trained and tested on their aseptic processing abilities, cleaner aseptic facilities are required, the quality of air entering the aseptic facility must be evaluated and maintained, sterilisation processes must be effective, knowledge of solution stability is needed and sterility testing of the products is required. The most common type of compounded sterile preparations (CSP) used clinically are aqueous injections. These CSPs require greater attention when being prepared as they pose the greatest risk to the patient if they are non-sterile or contain the wrong ingredients and/or wrong concentrations of ingredients if they are given intravenously. The main objective of sterile compounding is to prevent both morbidity and mortality of patients, which can be caused by non-sterility of preparations, high bacterial endotoxin content and errors associated with ingredients of the preparation, as mentioned earlier.
After the addition of the media, we insert an aeration tube inside and cover the lid with a cotton plug and start giving them aeration. This preparation has to be put on for 3 days under proper sunlight and 25-30 degree Celsius to observe if the culture is healthy/ potent or not depending on the color each culture portrays (The nanochloropsis culture should have a grass-green color to be seen as potent and the isochrysis culture should have a dark brown color to be seen as potent), if the colors seem dull and light, then that might mean that the culture is impotent.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Administration, U.S. Food and Drug. Animal and Veterinary. 02 Febuary 2014. Web. 17 Febuary 2014.
I prepared two large test tubes, each should have an inch of KOH pellets on the bottom of the tube. Next, a cotton ball is placed in each of the two test tubes above the KOH to plug the tube. Now one tube is filled to the top with peas, the peas are then removed and weighed to the nearest.1 grams, this is the experimental tube. The control tube is filled with plastic balls to the same height as the experimental tube. Next, a rubber stopper with attached capillary tubing is inserted in each test tube.
== § 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 = == = =
By using an inoculating loop, one loop of S41 is inoculated into 2 conical flasks of YEPA broth, YEPD broth and YEPPO broth respectively.