Name: Section: Due: January 26th, 2018 Worksheet 1: Isolation of Genomic DNA 1.
ID: 186082 • Letter: N
Question
Name: Section: Due: January 26th, 2018 Worksheet 1: Isolation of Genomic DNA 1. You isolate a sample of genomic DNA from Arabidopsis thaliana (a plant) and re-suspend (dissolve) the DNA in 6 ml of water. You make several dilutions of the DNA; then analyze the DNA by UV spectrophotometry. The limits of your spectrophotometer are 0.08-1.5. Below are your absorbance readings. Eill in the chart with the remaining values.(Show your work to gain partial credit in case of errors) Note: Think about how you would calculate the final concentration of your stock (6 ml) DNA tube from the concentrations of the individual dilutions Sample dH20 DNA Final Dilution A260 A280 Concentration Final A260/A280 Of Dilution Concentration (ug/ml) (ul) added Volume Factor of DNA Sample (Cug/ml) A 0 1000 1000 2.132 1376 B| 900 100 | 1000 | Le | 1.121 | 0.480 C 950 50 1000 0.550 0.230 1000 1 to 1 0.302 | 0.144 1000100 0.107 0.052 D 975 25 990 10 1000Explanation / Answer
Sample
dH2O (micro-liter)
DNA added (micro-liters)
Final volume (micro-liters)
Dilution factor
A260
A280
Con-
centration of Dilution (microgram
/ml)
Final Con-centration (microgram
/ml)
A260/A280
A
0
1000
1000
1
2.132
1.376
37.8
37.8
1.55
B
900
100
1000
10
1.121
0.48
32.05
320.5
2.34
C
950
50
1000
20
0.550
0.23
16
320
2.39
D
975
25
1000
40
0.302
0.144
7.9
316
2.1
E
990
10
1000
100
0.107
0.052
2.75
275
2.06
Nuclei acids DNA and RNA absorb Uv at 260 nm while aromatic amino acid in proteins absorb UV at 280 nm. A260 measure nucleic acids while A280 measures proteins and other contaminants. A260/A280 give an idea of the level of contamination in DNA sample. A value higher than indicates good quality.
A) Dilution Factor= Final volume/ Volume of DNA added=1000/1000= 1
A260 of 1.0 = 50µg/ml pure Double stranded DNA
Concentration of dilution (microgram/ ml)= (A260 reading – A320 reading) × 50µg/ml
= (2.132-1.376) X 50 microgram/ml= 37.8 microgram/ml
Final concentration (microgram/ml)= Concentration of dilution (microgram/ ml) X Dilution factor
= 37.8 X 1 = 37.8 microgram/ml
A260/A280 = 2.132/1.376= 1.55
The concentration is less because it is beyond 1.5 value of A260. The Uv spectrophometry sensitivity is 0.8-1.5.
Amount of DNA in tissue sample is 37.8 X 6= 728.07 micrograms
B) Dilution Factor= 1000/100=10
Concentration of dilution (microgram/ ml) = (1.121- 0.48) X 50 =32.05 microgram/ml
Final Concentration (microgram/ ml) = 32.05 X 10= 320.5 microgram /ml
A260/A280= 1.121/0.48= 2.34
Amount of DNA in orginal tissue sample - 320.5 X 6= 1923 micrograms
C) Dilution Factor= 1000/50=20
Concentration of dilution (microgram/ ml)= (0.550-.0.23) X 50= 16 microgram/ml
Final concentration (microgram/ml) = 16 X 20= 320 microgram/ml
A260/A280= 0.55/0.23= 2.39
Amount of DNA in original tissue sample= 320 X 6= 1920 microgram
D) Dilution Factor= 1000/25=40
Concentration of dilution (microgram/ ml)= (0.302-0.144) X 50= 7.9 microgram/ml
Final concentration (microgram/ml)= 7.9 X 40= 316 microgram/ml
A260/A280= 0.302/0.144= 2.1
Amount of DNA in original tissue sample= 316 X 6= 1896 microgram
E) Dilution Factor= 1000/10=100
Concentration of dilution (microgram/ ml)= (0.107-0.052) X 50= 2.75 microgram/ ml
Final concentration (microgram/ml)= 2.75 X 100= 275 microgram/ml
A260/A280= 0.107/0.052=2.06
Amount of DNA in original tissue sample= 275 X 6= 1656 micrograms
The concentration is less because it is less than 0.8 value for A260. The UV spectrophometer sensitivity is only 0.8-1.5.
Sample
dH2O (micro-liter)
DNA added (micro-liters)
Final volume (micro-liters)
Dilution factor
A260
A280
Con-
centration of Dilution (microgram
/ml)
Final Con-centration (microgram
/ml)
A260/A280
A
0
1000
1000
1
2.132
1.376
37.8
37.8
1.55
B
900
100
1000
10
1.121
0.48
32.05
320.5
2.34
C
950
50
1000
20
0.550
0.23
16
320
2.39
D
975
25
1000
40
0.302
0.144
7.9
316
2.1
E
990
10
1000
100
0.107
0.052
2.75
275
2.06
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