1) You will receive a DNA sample labelled A, B, C, or D. Make a 50-fold dilution
ID: 134521 • Letter: 1
Question
1) You will receive a DNA sample labelled A, B, C, or D. Make a 50-fold dilution of your sample and measure the absorbance at 260 nm. Calculate the concentration of your sample. Sample LabelB(o.052) 005 2 K 50 60ng/mL 130ng/mL 6cong DNA 2) A. Digest pul of your sample with 5 ??of Hind ? in 1X Buffer for a total digestion volume of 20 45 minutes at 37 °C uL. Add sample buffer for a final volume of 24 ?1. Incubate for l: S·iL 1 OX Hind III Buffer 5uLHind II Einzyme in IX Buffer AL Water uL Sample Buffer/ Loading Dye loXExplanation / Answer
1) formula for concentration of DNA in solution using absorbance at 260 nm :
(observed A260 ) x (Dilution factor) x 50 = DNA concentration in microgram/ ml
It has been standardized that 1 unit absorance of DNA at 260nm equals to approximately 50 microgram/ml of double stranded DNA
Here you diluted your DNA sample B 50 times. therefore dilution factor becomes 50. observed A260 is 0.052. Putting all these observation in the equation above
0.052 x 50 x 50 = 130 microgram/ml
2) your sample concentration is 130 ug/ml. you have to digest 600ng DNA from your sample .
using unitary method
130 ug/ml = 130 ng/ul
how: 1ug= 1000ng and 1ml = 1000ul
130 x 1000 ng / 1000 ul = 130ng/ul
now 130 ng DNA is present in 1 ul
1ng dna is present in 1/130 ul
600 ng dna is present in 600/130 ul which equals to 4.6 ul of DNA
Amount of Hind III enzyme to be taken is already mentioned i.e. 5 ul
Now for Hind III buffer: stock is 10X and working to made from it should be 1X. Apply simple M1 x V1= M2 x V2
10X x V1 = 1X x 20 ul
V1 = 2 ul
now for water subtract the volume of all the component i.e DNA, Hind III buffer, Hind III enzyme from total volume i.e 20 ul
20 - 4.6 -2 -5 = 8.6 ul of water
For loading dye the stock is of 6X concentration. Apply M1 x V1= M2 x V2
6X x V1 = 1X x 24 ul
V1 = 4 ul
Therefore 4 ul of loading dye will be taken
kindly check your calculation in 2nd question.
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