RNA in 100l of TE. To determine its concentration you mix 4 ul of the and take a
ID: 146007 • Letter: R
Question
RNA in 100l of TE. To determine its concentration you mix 4 ul of the and take an Aaso reading. What is the concentration of the mRNA sample if the A2eo reading you obtain is 0.027? (2pts) 2. How much mRNA did you isolate? Show calculation. (2pts) 3. An Aeo reading for this sample was 0.018. Do you tell your boss that you have just isolated 'excellent quality mRNA? Why or why not? (1pt) 4. You typically use 0.4g Of NHindill ladder when you run an agarose gel. Calculate what % each band is of the total DNA loaded. Calculate the amount of DNA (vg) each band represents. (5pts) 23,130 bp band: 9,419 bp band: 6,557 bp band: 4,371 bp band: 2,322 bp band: 2,028 bp band: 564 bp band: 125 bp band: % of Total Amt of DNAExplanation / Answer
1. A spectrophotometer is used to measure the concentration of DNA/RNA. The concentration of nucleic acid is measured using Lambert-Beer law which states that there is a linear relationship between absorbance and the concentration of the sample. Based on that DNA or RNA absorbs at 260 or 280nm of light. The optical density of 1.0 at 260 nm is shown below for various solutions.
- 50ug/ml of ds DNA
-40ug/ml of RNA
-33ug/ml of ss DNA.
so the formula for calculating the RNA conc. is = O.D260 X 40ug/ml X dilution factor. (Dilution factor Vfinal/Vinitial)
= 0.027 X 40ug/ml X 250
Concentration = 270ug/ml
2. We have a total of 100ul of RNA and the concentration of RNA is 270ug/ml
In 1000ul we have 270 ug of RNA
So, in 100ul we will have 27ug of RNA.
3. The quality of RNA is decided on the basis of O.D260/O.D280. Good RNA has an O.D260/O.D280 of 2.0. In this case, the 260/280 ratio is 1.5. Here, the RNA is not of good quality a value of less than 2 suggests that there is contamination of proteins, phenols etc which absorbs strongly at 280nm.
4. You can calculate the % of DNA and the molecular weight if you know the total amount of DNA loaded. Here it is given that 0.4ug is loaded. To calculate you should know the band sizes of the DNA ladder. First, calculate the total base pair sum of the DNA band and for that sum up all the band sizes.
total base pairs = 48516
the top band is 23130 bases represents 23130/48516= 0.4767 =47.7 % of DNA
and the amount of DNA will be 0.4767 X 400ng = 190.4 ng
similarly, you can calculate for the rest
9419bp = 19.4 % = 77.6ng
6557bp = 13.5% = 54ng
4371bp = 9% = 36.03ng
2,322bp = 4.8% = 19.14ng
2,028bp = 4.2% = 16.72ng
564bp = 1.1% = 4.65 ng
125bp = 0.3% = 1.03 ng
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