Unit 8 Ml Lab Report

• Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.

Discard the solution in the appropriate container as directed to you by your lab instructor.

Using the graduated cylinder, measure 20mLs of the stock sucrose solution and 180mL of water to create a 3% sucrose solution and place it into the 250mL beaker (beaker #2). Place bags #1‐3 (red, blue, yellow) into beaker 2 and bag #4 (green) into beaker 1. Allow the bags to sit for one hour. After allowing the bags to sit for one hour, remove them from the beakers carefully open the bags, noting that often times the tops may need to be cut as they tend to dry out. Measure the solution volumes of each dialysis bag using the empty 250 ml beaker.

Fill a test tube about 1/3 full with cold tap water for use in step 34.

The first experiment begun by filling a 600-ml beaker, almost to the top, with water. Next, a 10-ml graduated cylinder was filled to the top with water. Once water was added to the beaker and graduated cylinder, a thumb was placed over the top of the graduated cylinder. This would ensure that no water was let out and no bubbles were let into the graduated cylinder. Next, it was turned upside down and fully submerged into the beaker. Then, a U-shaped glass tube was attained. The short end of the glass tube was placed into the beaker with the tip inside of the graduated cylinder. Next, a 50-ml Erlenmeyer flask was received. After, 10-ml of substrate concentration and 10-ml of catalase/buffer solution were placed into the flask. A rubber stopper was then placed on the opening of the flask. After adding these, the flask was held at the neck and spun softly

To make the buffer solution you need 0.2 mol dm-3 of Na2HPO4 and 0.1mol dm-3 of citric acid this will give 100cm3 of buffer. Here is how to get the different pH in the buffer solutions:

XII. Take the 250 ml beaker to your lab bench. Set up a gravity filtration with a plastic funnel, folded wet filter paper, and an Erlenmeyer flask. Pour the content in the 250 ml beaker slowly through the filter paper. Wash the filter paper with deionized water. Dispose of the filtrate in the proper labeled waste container.

1.) Transfer the distillate to separatory funnel. Fluid didn’t seem very clear but sufficient to finish our lab on time.

The students selected eluting flow through, E.coli flow through, and washing flow through samples from lab #6 that contained protein. For all samples, they mix 15 microliters of the selected sample with 4 microliters of 5x sample loading buffer and 1 microliter 20x reducing agent, and heat the samples at over 90 degree Celsius for 5 minutes. In the meantime, the students proceed to prepare 40 ml of 15% resolving gel solution using 9.68 ml of H2O, 20 ml of 30% Acrylamide/bis solution, 10 ml of 4 x resolving gel buffer, 300 ul of 10% APS, and 20 ul of TEMED. First they prepared the resolving gel buffer, to do this they mix 9.09g of 1.5 M Tris-HCl, 0.2g of 4% SDS, and approximately 30 ml of H2O. When all the components where mix, they checked the pH and verify that the solution has a pH of 8.8, if that was not the case they proceed to add HCl or Tris, depending whether the solution was more acidic or base, until the desire pH was reach.

Pour 10 ml of the affinity gel into the column. Allow a few minutes for the solution to harden and transform into a gel-like consistency.

Submerge the graduated cylinder in the plastic tub so that it is completely filled with water. Hold the open end of the graduated cylinder and move it vertically upside-down where the open end of the graduated cylinder is still submerged in the plastic tub. Clamp the graduated cylinder the ring stand of the lab table to keep it in place. perforate a hole in the top of the rubber cork for the solution container. Cut a straw the length of about four inches. place the straw inside of the rubber cork hole. Set up your timer for two minutes.

10 µL of 2x SDS sample buffer was added to the 10 µL of provided unknown protein sample.

3. Use a sterile pipette to transfer 0.1 ml of each dilution on to a MacConkey agar plate.

Then added 200 ul of P1 Buffer (Red) to the tube and re-suspend pellet completely. We then centrifuged at 11 rpm for 1 min and discarded the supernatant. Then we added 200 ul of P2 Buffer (Green) and mix by inverting the tube 2 - 4 times. Cells are completely lysed when the solution appears clear, purple, and viscous. (Proceed to the next step within 1-2 minutes) We then centrifuged at 11 rpm for 1 min and discarded the supernatant. We added 400 ul of P3 Buffer (Yellow) and mix gently but thoroughly. The sample will turn yellow when the neutralization is complete then incubate the lysate at room temperature for 1-2 minutes. We then centrifuged at 11 rpm for 2 min and transferred the supernatant to a Zymo-Spin column. Once transferred we spun for 30 seconds at 11,00 rpm and then spun with 200 ul of Endo-wash for 30 seconds then 400 ul of plasmid wash buffer for a minute. After that we spun our column empty for an additional minute to remove an excess alcohol. We then transferred out column to a clean centrifuge tube and added 30 ul of Elution Buffer to the column and incubated at room temperature for a minute and centrifuged for 30 seconds. The result was our plasmid DNA.

All PN solutions are changed at 24 hours. PN solutions containing dextrose and amino acids