Question
please calculate the volume needed in #4
Ul lInBchondrial ('Petite') Mutants of S. cerevisiae A-intercalating agent ethidium bromide (EiBr, Mw-394.3) selectively affects tites" that form smaller colonies than wild-type cells. We will induce the cells called "pet formation of petites by EtBr mutagenesis and analyze the deficiencies associated with the loss of mitochondrial DNA in the following experiment. 1. An overnight culture of CTY10-5d yeast in YPD medium will be grown for each class. The CTY10-5d strain of S. cerevisiae is deficient in a gene of the adenine biosynthetic pathway. In the absence of supplemental adenine, they grow more slowly than true wild-type S: cerevisiae and develop a red pigmentation. 2. Aliquots (about 2 mL) of the yeast culture will be provided to each group to use in determining the OD60o of the culture: A. Make dilutions of 15 and 110 in non-sterile YPD media. B. Blank the Spectronic 20 with non-sterile YPD media. C. Record the ODeoo for both of your dilutions For this S. cerevisiae strain, 1 OD60 2 x 10' cells/mL. (How close did the calibration curve you generated using the ODeoo and hemacytometer data from 2014 [Microscopy and Cell Counting" problem set] come to giving this value?) D. Calculate the ODso and the concentration (cells/mL) of the undiluted culture: 3. Two culture tubes containing exactly 3 mL of sterile YPD ands a separate, sterile aliquot of the yeast culture will be provided to each group. A. Using sterile technique, dilute the overnight yeast culture 1:100 into each tube. (Note: B. Label tubes with your initials and a different dilution may be recommended in class; record the actual dilution used!) - for the control culture + for the EtBr-treated culture 4. Add EtBr from a sterile 0.1% (w/v) stock solution to the "+" culture to a final concentration of 20 g/mL. Before class, determine the volume of stock EtBr required. For your lab report, you will need to convert this concentration to M (see MW provided above). Add an equal volume of sterile ddH20 to the"" culture. Provide your calculations: Mutagenesis-1
Explanation / Answer
A 0.1% EtBr solution will mean that 100 mL solution contains 0.1 g EtBr.
We know that 1 g = 1000 mg; therefore, 0.1 g = (0.1 g)*(1000 mg/1 g) = 100 mg.
Therefore, we have a stock 100 mg/100 mL = 1 mg/mL solution. The final concentration desired is 20 µg/mL = (20 µg/mL)*(1 mg/1000 µg) = 0.02 mg/mL.
The dilution factor is DF = (1 mg/mL)/(0.02 mg/mL) = 50 (ans).
