prepare your DNA concentration graph, you will need to plot the absorbance at 26
ID: 175316 • Letter: P
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prepare your DNA concentration graph, you will need to plot the absorbance at 260nm versus concentration of DNA in the standard sample. You wil then need to perform linear regression lysis (see document on eLeam for instructions) to get the equation of the line. This equation can be used to solve for the DNA concentration in your samples since you know the absorbance our DNA samples you can solve for their concentration X-value) (y-value) the DNA purity plot you will need to take the 260nm absorbance values and divide them by the nm absorbance values; these will be the y values in your plot. Then you need to calculate the protein in samples C, D, E. F. and G. The protein in the sample can be calculated using the owing equation: protein Protein protein-DNA x100. These wil be the x-values for your plot. Once have these values plotted, perform a linear regression analysis and you can use the orbance value to solve for the percent protein in your DNA samples. a Analysis: 1. For the DNA Purity calibration curve you constructed, calculate the equation for the curve (see the Linear Regression Analysis document in eLeam for help) 2. The students in PerfectWorld isolated their DNA (with and without the addition of pineapple juice) and obtained UV absorbance data at both 260 and 280 nm. That data is shown below. Use your calibration curve to calculate the purity of the DNA preparations obtained in PerfectWorld Sample (a) Pineapple Juice LAbsorbance 260 nm) Absorbance (280 nm) 0.72 0.44 0.55 3 Yes 097 0.57 4 072 0.58 5 No 090 071 6 No 097 0.275 3. Why did you determine absorbance at 260 and 280 nm when determining DNA purity? What biomolecules contribute to absorbance at these two wavelengths? What specific chemical entities contribute to each absorbance? 4. All of the PerfectWorld samples contained 50ug/mL DNA. Why is the 260nm absorbance not the same for all the samples? 5. What is the purpose of the pineapple juice? Canned pineapple juice will not work in this in the purity of the PerfectWorld DNA preparation (if any) with and without the pineapple juice fwhat are the calculated values, means t std dev)? 6. An extinction coefficient for double stranded DNA is 0.02 uUIng cmp. Which preparation would you be comfortable using the 260nm absorbance and this extinction coefficient to calculate the concentration of DNA from? What is that concentration in ug/mL? Why would you be uncomfortable calculating a concentration for the other sample? 7. What does the detergent do in this procedure 8. Why is it critical not to heat the banana solution over 70 C? What would happen to your yield of DNA and why? dified from "Genomic DNA Purification Student Laboratory Manual", prepared by PromegaExplanation / Answer
Answer :
Q5. The Pineapple juice consisits act as tendersier which is mostly used in meat industry for the purpose of meat tenderisation .
The pineapple jucie consists of two enzymes namely Bromelain and papain which acts on the DNA to remove the proteins bound around the DNA molecule.
The pineapple juice which is canned has lost hte ezyme activity in the pineapple juice due to sterilization duing the canning. the optimum temperature for the enzyme to work is 370C.Hence canned pinapple juice is not used.
The Difference between the perfect DNA world preparation is the purity of the DNA obtained due to the activity of enzymes Bromelin and papain which removes the histones and proteins which are wound around the DNA.
The calculated values for the above measured samples are as follows
For Sample 1 the 1.63 absorbance which is less than 1.8 (The dna which is showing 1.8 ratio,the absorbace of pure DNA less than that shows the contamination with Histones or proteins. above 1.8 indicates the sample is conataminated with RNA)
Q.6 Molar extinction coefficent
Single stranded DNA has the comfortable when using the value given the chemistry lecturers as the single stranded DNA absorb more of Calculation at 260.
The extinction coefficient is the absorbance divided by the concentration and the pathlength, according to Beer's Law (epsilon = absorbance/concentration/pathlength). The units of extinction coefficients are usually M-1cm-1, but for proteins it is often more convenient to use (mg/ml)-1cm-1. If the molecular mass of the protein is known, divide mg/ml (-g/L) by mass in g/mole to get mole/L.
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