Question

Hi I am im a biochemistry lab and we are doing an experiment titled: PCR Amplification of LeuRS F C Terminal Domain. I am having troubles figuring out the rest of my caluclations.

If the average of your weights pipettes, or more than 5% off for the pipet, what is the average value of your masse your weights are more than 2.5% off from the theoretical values for the P1000 or P wan 5% off for the P20 or P10 pipettes continue practicing your technique. Relative to th the average value of your masses reflect? Please answer below. If individual readings vary more than 1% from o correctly and/or if your pipette leaks. In this case, w vary more than 1% from one another check to make sure you are operating the pipett your pipette leaks. In this case. what are we evaluating in terms of your pipetting technique? Please answer below. To check for leaks draw up the maximum volume, hold the pipe volume. hold the pipette in a vertical position (tip pointed down and son lauld drive ut during the first 4 seconds. If it is dripping make a note, and let Dr. Weitzel know so that he can get the seals and O-rings replaced EXERCISE ON DILUTIONS Obtain one 1.5 mL aliquot of a 9 ma/ml bovine senum albumen (BSA) solution. From this solution you will prepare two separate sets of dilutions. You will also need twelve (12) 1.5-ml microfuge tubes. Dilution set 1 Prepare the following dilutions of the 9 mg/ml BSA stock solution in distilled water: a) 1/5 b) 1120. Prepare each dilution in duplicate (independently from each other in 6 separate microfuge tubes. The final volume in each case should be 1.0 ml. Be sure to label each tube appropriately (e.g. "5X-1a" and "5X-1b" for the duplicates of the 5-fold dilution of Set 1, etc). Please write your calculations on the back of this worksheet. Dilution set 2 Prepare a second set of the same dilutions of the BSA stock solution in distilled water, a) 1/5; b) 1/20; c) 1/100 In this case, however, you should prepare them as serial dilutions. This means that you will prepare the 5-fold dilution using the 9 mg/ml stock, and the 20-fold dilution using the 5-fold dilution, etc. Prepare two separate series in the dilution set. Be sure to label each tube appropriately so as to distinguish them from Dilution Set 1 (e.g. "5X-2а" and "5X-2b"). Please write your calculations on the back of this worksheet. READING ABSORBANCE You will check your dilutions by reading the absorbance in the spectrophotometers. We want to use absorbance to confirm that the dilutions were performed correctly. 1. Remove the protective cover from the spectrophotometer and turn it on by pressing the toggle switch in the back. The power-on sequence will appear on the display and the instrument will go through its self- diagnostics. The lamp needs to warm up for 30 minutes before taking any readings. 2. Place a 1-ml volume of distilled water in a disposable UV cuvette. Wipe the cuvette with a kimwipe to remove any fingerprints. Place the cuvette in the bench-top spectrophotometer with the arrow facing you. It is essential that you use the correct type of cuvette and that it is correctly oriented in the spectrophotometer. 3. Press "Measure Blank" to blank the instrument. When the instrument is finished measuring the blank, the message disappears. 4. Remove and empty the cuvette. Add the entire volume of one of the 1/5 dilution in Set 1. Wipe the cuvette with a kimwipe. 5. Place the cuvette in the holder and allow the reading to stabilize. Record the value in the table below. 6. Continue reading the absorbance of each dilution, being sure to rinse the cuvette with distilled water between each reading. Wipe the cuvette with a kimwipe prior to each reading. Record all values in the appropriate box of the table below.

I already did the calculations for the first dilution set, I was wondering how I would do the calculation for the second dilution set.

Also I was wondering how I would determine the theoretical values for my data. Is there a specific formula I can use?

Answer 1

They are asking to do the dilutions in serial way. That is: you have to do the "smallest" dilution (1/5) from the standard solution, the following (1/20) from the 1/5 and the following (1/100) from the 1/20. Serial dilutions would be something like "diluting the previous dilution".

So now, the calculations:

• The 1/5 dilution from the standard solution is exactly the same as in the first dilution set: 200 mL of BSA and 800 mL of water. Now, using the dilution formula, we can calculate the concentration in this solution:

Vi*C C2 = - 200mL * 9mg/mL = 1.8mg/ml 1000mL V

• Now, for the 1/20 dilution, we can calculate the concentration it should have, which is 1/20 of the original concentration. That is: 9/20 = 0.45 mg/mL. Now we have to calculate the volume of dilution 1/5 we need to take to prepare this. Dilution 1/5 has a concentration of 1.8 mg/mL and we need a final volume of 1 mL. We use the same dilution formula to calculate V₁ (the volume of 1/5 dilution):

Vi=- V2 * C2 C 1000mL *0.45mg/mL = 250mL 1.8mg/mL

We have to take 250 mL of the 1/5 dilution to prepare the 1/20 dilution.

• Now it's the same thing, but we have to prepare de 1/100 dilution (which is equivalent to a concentration 0f 0.09 mg/mL) from the 1/20 dilution (which has a concentration of 0.45 mg/mL):

V2 * C2 Vi=- 1000mL * 0.09mg/mL = 200mL 0.45mg/ml C1

We need 200 mL from the 1/20 dilution to obtain de 1/100 dilution.

To calculate the theoretical data of absorbance there is in fact a formula:

Abs=s* *

Where epsilon is the given value of 0.667 (mg/mL)^-1 cm^-1, l is the optical path, which is usually 1 cm for spectrophotometers and c is the concentration. Thus, by multiplying the calculated concentrations by 0.667, yo will get the theoretical absorbance values.