Question

You receive 12 samples in your laboratory for genetic screening. The samples form two families. The first family is composed of samples: 1(Mom Smith); 2 (Smith Son 1); 3(Smith Daughter 1); 4(Smith Son 2); 5(Smith Son 3); 6(Smith Son 4); 7(Smith Son 5); 8(Smith Dad) and 9(Mom Jones); 10&11(Jones twin boys); 12(Jones Dad). Mom Smith and Dad Jones are siblings.

Given the following spectrophotometric readings calculate the purity and concentration of the stock solutions.

Desired Ranges:
260/280=1.8-2.0
260/250=1.0-1.2
230/260=0.4-0.5

5uls of stock DNA were diluted with 995 HOH and measured.

Sample	A280	A260	A250	A230	260/280	260/250	230/260	ug/ul of stock
1	0.189	0.350	0.313	0.151
2	0.054	0.095	0.084	0.041
3	0.089	0.159	0.142	0.072
4	0.111	0.201	0.180	0.092
5	0.130	0.237	0.212	0.108
6	0.128	0.231	0.206	0.102
7	0.032	0.048	0.043	0.029
8	0.057	0.101	0.090	0.046
9	0.141	0.256	0.230	0.117
10	0.122	0.220	0.196	0.098
11	0.086	0.151	0.135	0.072
12	0.149	0.270	0.242	0.134

Based on the above information, do you predict any difficulties in performing a restriction digest on any of the above samples? Explain

Based on the information calculated previously set up a digest with each sample that contains 10ugs of DNA/sample and 40 units of Hae III enzyme.

Sample	uls DNA	uls 10XBuffer	uls HOH	uls Enzyme	Total
1
2
3
4
5
6
7
8
9
10
11
12

Answer 1

Sample	A280	A260	A250	A230	A260/A280	A260/A250	A230/A260	ug/ml of stock
1	0.189	0.350	0.313	0.151	1.8518519	1.1182109	0.43142857	17.5
2	0.054	0.095	0.084	0.041	1.7592593	1.1309524	0.43157895	4.75
3	0.089	0.159	0.142	0.072	1.7865169	1.1197183	0.45283019	7.95
4	0.111	0.201	0.180	0.092	1.8108108	1.1166667	0.45771144	10.05
5	0.130	0.237	0.212	0.108	1.8230769	1.1179245	0.4556962	11.85
6	0.128	0.231	0.206	0.102	1.8046875	1.1213592	0.44155844	11.55
7	0.032	0.048	0.043	0.029	1.5	1.1162791	0.60416667	2.4
8	0.057	0.101	0.090	0.046	1.7719298	1.1222222	0.45544554	5.05
9	0.141	0.256	0.230	0.117	1.8156028	1.1130435	0.45703125	12.8
10	0.122	0.220	0.196	0.098	1.8032787	1.122449	0.44545455	11
11	0.086	0.151	0.135	0.072	1.755814	1.1185185	0.47682119	7.55
12	0.149	0.270	0.242	0.134	1.8120805	1.1157025	0.4962963	13.5

The DNA ration at 260/280=1.8-2.0 shows the purity of DNA samples, This purity of DNA is required for restriction digestion experiment. The sample No 1, 2, 7, 8 and 11 are showed less that 1.8 OD ratio which indicates the DNA is not pure. Hence these DNA samples can not be used for restriction digestion experiment. Since these samples revealed proteins contamination as their OD is less than 1.8 OD ratio at A260/A280nm. when such a DNA us used for the restriction digestion, they doesn't generate proper restriction digestion pattern as DNA itself not pure.