13 Jun BIOL 1362 How do you prepare a sterile 10 mL of 100 mg/mL
Question
Problems:
1. How do you prepare a sterile 10 mL of 100 mg/mL ampicillin and where do you have to store the stock solution?
2. Let’s say you are TA for biotechnology class. Your professor asked you to prepare 100 LB agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% bacto-agar) plates containing 100 µg/mL of ampicillin. You assume each Petri plate needs 25 mL, and you have 100 mg/mL ampicillin stock solution. Describe in detail where and how to prepare the plates including equipments to be used.
3. You are doing a large scale- plasmid DNA extraction from E, coli culture. Let’s say the protocol called for your cell suspension to have a concentration of lysozyme at 2 mg/mL. Your suspension has a total volume of 10 mL. The lysozyme stock tube has a concentration of 20 mg/mL. How much lysozyme should you add?
4. You have already determined the doubling time of 24 min for a certain E. coli strain. You also determined the cell density of 107 cells/mL by plate counting. You diluted the cell to 1:100, inoculated 0.1 mL of the diluted cells into 9.9 mL of the same medium, and incubated under the same growth condition that was used to determine the doubling time and cell density. How long should you incubate to reach a cell density of 32,000 cells/mL?
5. Let’s say the A600 of the 5 mL overnight culture at 10:00 AM is 1.0 and you want to harvest 10 mL culture of E. coli cells at A600 = 0.8 at 2:00 PM on the same day. You know E. coli doubles every 30 min under the growth conditions used. How much volume of the 5 mL culture would you need to inoculate into a fresh 10 mL medium? Assume that only 50% of cells in 5 mL overnight culture is alive.
6. Calculate the theoretical total DNA yield in (µg) and DNA concentration (ng/µL) assuming that you recover 100% of the pure E. coli genomic DNA (4.6 x 106 bp) in 0.1 mL of final DNA sample from a 2-mL overnight culture of 2 X 109 cells/mL. MW of DNA base pair is 660 g/mole.
7. Absorbance A570 of an unknown BPB solution was found to be 0.445. Calculate concentration in mg/mL of the unknown BPB (MW = 670) solution using the standard calibration curve a below.
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8. The human genome is 3 x 109 bp. The MW of 1-bp dsDNA = 660 g/mole If you have 1 microgram of human genomic DNA, how many copies of the genome do you have?
9. Your DNA concentration is 10 mg/mL. How many pg/µL is this?
10.The spectrometric readings at A260 of 1:100 diluted DNA sample is 0.15. What is the DNA concentration before dilution?
11.The spectrometric readings at A260 of 1:100 diluted RNA sample is 0.25. What is the RNA concentration before dilution?
12.To quantitate a target RNA present in a cell, competitive RT-PCR was performed by placing constant amount (10 ng) of total RNA and increasing amounts of competitor mRNA (0. 17, 50, 150, 450, and 1350 pg) into six PCR tubes containing RT-PCR master mix and the same set of primers. RT-PCR of target and competitor RNA produces 750 and 700 bp DNA fragment, respectively. The synthetic competitor DNA with virtually same GC content and sequences as those of target DNA was obtained from in vitro transcription of cloned plasmid DNA. After PCR, the reaction products were run onto agarose gel, and the gel was stained with GelRed dye. Fluorescent bands were visualized under UV light, and the intensity of each band in a gel photograph was quantitated by ImageJ scan analysis. The obtained results are shown below. Based on the scanning density data, calculate the quantity of target mRNA present in 10 ng of total RNA isolated from cells. T1 T2 T3 T4 T5 T6 C1 C2 C3 C4 C5 C6 Density 22.52 17.26 5.31 2.62 1.75 0.68 – 2.18 4.08 9.88 14.63 19.09
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13.How would you prepare if you want to prepare 104 copies of a 272-bp ds DNA molecule for a copy number standard of PCR using the stock DNA solution of 1 mg/mL? MW of a 1-bp dsDNA is 660 g/mole.
14.You must make 1 L of 0.2 M of acetic acid. All you have available is concentrated glacial acetic acid (assay value 98%, specific gravity is 1.05). How many milliliters are needed to make this solution?
15.Recombinant plasmid (vector + insert) was transformed into yeast for amplification. The yeast genome contains 12,495,682 base pairs. A plate can hold approximately 200 transformants. If the average insert size was 2 kb, how many plates do you have to make in order to have a 99% probability of getting the correct fragment carrying a target gene of interest?
16.When target DNA fragment reaches 1012 molecules, its amplification efficiency drops dramatically and stops accumulating PCR fragment, reaching the plateau phase. You are to PCR-amplify a 580-bp target DNA from a 14-kb plasmid DNA template.
(a) What amount of template DNA should you use to produce 1012 copies of the target DNA molecules after 20 cycles, assuming that overall amplification efficiency is 80%? The molar mass of 1-bp dsDNA is 660 g/mole.
(b) How would you dilute the template DNA in order to add 2 µL of the diluted DNA to PCR reaction mix if the concentration of DNA template is 0.61 µg/µL?
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