1. Label each well of a tissue culture treated 6-well plate appropriately for each cell line or condition being investigated. 2. Prepare 2x cell culture medium by dissolving 1 g of powder medium and 0.2 g of sodium bicarbonate in de-ionized water to a final volume of 50 ml. 3. Pass this medium through a 0.2 μm filter to sterilize. 4. Add additional components needed for normal culture of the cell line of interest. For example, grow CMT 167 cell line in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin solution. Warm medium to 37 °C in hot water bath prior to use. 5. Prepare 1x cell culture medium separately as you would for normal cell culture of the cell line of interest. 6. Prepare 1% noble agar by adding 1 g of noble …show more content…
Autoclave the noble agar mixtures to sterilize. These mixtures can be made in advance and stored at 4 °C but should be heated again at the time of the experiment until agar has completely dissolved. 9. Prepare nitroblue tetrazolium chloride solution by making a 1 mg/ml stock solution in 1x PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in H2O to final volume of 1,000 ml). This will be used at the end of the experiment to stain the colonies. Plating of Bottom Layer of Agar: 1. Loosen the cap on the bottle of 1% noble agar and microwave for about 1-2 min. While heating in a microwave, monitor the solution closely to avoid boiling over. Continue heating, while mixing intermittently, until agar is completely dissolved and the solution is clear. NOTE: Use heat-resistant gloves to handle flask after heating. Failing to do so may cause burn or serious injury. 2. Place melted agar solution and pre-warmed 2x culture medium in an ice bucket filled with hot tap water (42 °C). Also place a 50 ml conical tube in a tube holder in the ice bucket with hot water. Transfer bucket to cell culture hood for subsequent steps. 3. For the bottom layer of agar, you will need 1.5 ml of a mix of agar and medium per well of a 6-well …show more content…
First, harvest cells by trypsinization and dilute them 1:5 in culture medium (e.g. for 1 ml of trypsin, add 4 ml of medium) into a 15 ml conical tube. 3. Count cells and calculate the number of cells needed per well to prepare a final cell suspension at this time. This number will vary depending on cell type. Use 5,000 cells/well as a starting point and adjust as needed. For this cell number, you would prepare a cell suspension of 6,667 cells/ml (i.e. each well will receive 0.75 ml of this suspension and 0.75 ml of agar for a total volume of 1.5 ml; the concentration of cells will also be diluted 1:2 for a final total cell count of 5,000). 4. The volume of cell suspension needed per well of a 6-well plate will again be 1.5 ml. Prepare additional cell suspension totaling 12 ml per 6- well plate. 5. Melt 0.6% agar solution in a microwave as above and place into ice bucket containing hot water along with a 50 ml conical tube in a tube holder and the final cell suspension from Step 3. 6. Transfer the ice bucket with melted 0.6% agar to cell culture hood for subsequent steps. 7. Mix 0.6% agar and cell suspension in a 1:1 ratio, preparing a total volume of 12 ml per 6-well plate. 1.5 ml will be required per well but extra should be made as
This is what I will mix with the varying amounts of Strontium nitrate to form the precipitate. • Strontium Nitrate (enough to fill 8 boiling tubes with your varying amounts/36cm³) This is what I will mix with the sodium carbonate in order to form my precipitate. • Test Tube Rack
Then they are shocked with a calcium chloride solution that changes the charge on the cell membrane so that the plasmid DNA may be accepted into the cell. This solution must be chilled so that the cell membrane may heal. After incubating the bacterial cells, they are heat shocked to open the pores in the cell membrane to allow the transformation to occur. After being chilled again in order not to melt the agar, the cells are placed in a medium
Using two separate aseptic pipettes, 250 µl of LB broth were added to each micro test tube and mixed gently. Likewise, using two separate, aseptic pipettes for each tube, 100 µl of solution was added to the appropriate agar plate. After, using a new loop for each plate, the solution was spread gently across their surfaces. Lastly, the plates were stacked, taped together, and labelled before placing them upside down in an incubator set at 37°C
One needs to be labeled +PGlo and the other container is labeled -Pglo. Next, in both test tubes using a dropping, 250 microliters of calcium chloride is needed (Making sure to use a new dropper after each test tube). Then place both containers into a styrofoam cup containing crushed ice a. Taking a sterile loop, scrap a thin layer of E.coli from the petri dish onto the loop (making sure the loop gets a clear film over it). Remove the +pGlo test tube and open the container. Place the loop with the nonpathogenic E.coli and twirl it around in the test tube.
After its melted and you don’t see any clumps you pour it into the mold to cast the gel and wait 20-30 minutes for the gel to set. Plug in your electrophoresis system and assemble the platform in the tank. After the gel sets remove it from the casting stand, setting the gel tray inside the tank on the platform making sure the wells are facing the negative side. Measure out 125 ml of diluted SB running buffer and pour slowly into one side of the tank, removing all air bubbles. Turn on the low intensity light and load your DNA samples using 10 μl per well, changing your pipette tip after every sample.
Tube 1 had 1 drop, tube 2 had 2, and each tube after had an additional drop until tube 5. Next, deionized water was placed in each tube. Tube one had 4 drops; tube 2 had 3 drops and the pattern continued until tube 5. After each tube was filled with the glucose and deionized water, the contents were mixed and centrifuged. After the tubes were centrifuged, any pellets formed during the process were removed.
3. With the cheek cell we had to put a drop of methylene blue on top of the smeared sample on the slide we collected, so the animal cell nucleus would be more visible under the microscope and then put the slide on again starting at a 45-degree
Placed the four test tubes into foam microtube holder and placed in water bath to incubate at 37 degrees Celsius for 45 minutes. Ms. Lovrien added 5 L of 10x loading solution to tubes labeled 1-4. Placed samples in the refrigerator overnight. After returning to class the next day, obtained a gel tray that contained 0.8% agarose with TAE buffer.
Observations: 1. The first step had to be repeated due to not following proper instructions. I did not grease the screw, so as I was shaking the mixture, solids were forming around the screwpart of the separatory funnel. 2. When adding 5.0 mL of NaOH to the unknown mixture and shaking it for about 30 seconds, layers had formed.
= 10^-3 M = 1,000 mL Here C1,C2; are the first and second concentrations of solution V1 and V2 ; are the required and current volumes. The impeller turned on and DDA, and tap water left to be mixed properly with water for 2 minutes. Approximately 150 grams of quartz added into the solution.
Fill the well with 90ml dh20 to reach 100ml. move 10 ml of the second well to the third well. FIll the third with another 90ml dh20 to reach 100ml. Move 10 ml of the third well to the fourth well. Fill the fourth well with 90ml dh20 to reach 100ml.
From the Unknown tube professor Cooper gave me, I scratched a little on the slant surface with the sterilized inoculating loop. Then I place it on a clean prepared slide which already had a slight drop of water. The two substances are mixed together in the middle of the slide and let dry completely. One extra step of “heat fix” is necessary to adhere everything to the surface of the slide. To start gram staining, I slightly pour crystal-violet all over the slide and let it sit for 30 seconds before wash it off with water.
The animal blood cells were examined first. There were three sample tubes, labeled 1-3, set up with 0.1 ml of mammal blood. First, we only added 1.00 ml of Stock 300 mM NaCl to
It also better to ensure that the materials like hemocytometer and pipettes are sterilized and clean so that there would be lesser or no artifacts would be seen under the microscope. The researcher was to use trypan blue exclusion method to test for cell viability, observe the non-viable and viable cells, and was able to have a cell count using the
Escherichia coli are transferred into the medium through an inoculating loop and are later slide from the bottom to the top forming a straight line. The bacteria appear to have effused as it spreads outwards. This shows that Escherichia coli are not motile as it only spread out from the straight line. The third method is stab culture. Escherichia coli and Bacillus subtilis are grown by stabbing vertically into nutrient gelatine in a test tube.