User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/10

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Nuclear Gene Adenita Culture 454 Amplification

Re-Amp Instagene Cultures 57, C, D, I

EX Takara RXN Mixture conc 1X 5 ' '
Milli Q water 4.15 20.75
Takara EX Buffer 10X 1 5
dNTP 2.5uM 0.8 4
Primer Mix 20uM 1
EX Taq 5U 0.05 0.25
Template Instagene 3 15
45
9.0ul / RXN add 1.0ul Primer
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Primer AMP Lane Template Primer
1 Instagene 57 B3 16 Instagene I B3
2 Instagene 57 C3 17 Instagene I C3
3 Instagene 57 D3 18 Instagene I D3
4 Instagene 57 E3 19 Instagene I E3
5 Instagene 57 F3 20 Instagene I F3
6 Instagene C B3 26 Neg Control B3
7 Instagene C C3 27 Neg Control C3
8 Instagene C D3 28 Neg Control D3
9 Instagene C E3 29 Neg Control E3
10 Instagene C F3 30 Neg Control F3
11 Instagene D B3 31 Pos Control B3
12 Instagene D C3 32 Pos Control C3
13 Instagene D D3 33 Pos Control D3
14 Instagene D E3 34 Pos Control E3
15 Instagene D F3 35 Pos Control F3
*Positive Control = AV 20ng

Load 2.0ul Sample

10June10.jpg

Nuclear Gene Adenita Culture 454 Amplification QIAquick Cleaning

1) Combined products from primer 2-6 for each culture 2) Cleaned with Qiagen QIAquick PCR product purification kit

http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2

see pg 19

Nanodrop results

Sample ID Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
1 75.2 ng/µl 1.504 0.823 1.83 2.15 DNA 50
2 60.5 ng/µl 1.211 0.665 1.82 2.17 DNA 50
3 90.6 ng/µl 1.812 1.014 1.79 2.2 DNA 50
4 97.2 ng/µl 1.945 1.071 1.82 2.18 DNA 50
5 52.8 ng/µl 1.055 0.584 1.81 2.14 DNA 50
6 82.6 ng/µl 1.653 0.892 1.85 2.07 DNA 50
7 75.3 ng/µl 1.506 0.813 1.85 2.08 DNA 50
8 56.5 ng/µl 1.129 0.634 1.78 2.15 DNA 50
9 87.5 ng/µl 1.75 0.953 1.84 2.23 DNA 50
10 65.2 ng/µl 1.305 0.725 1.8 2.09 DNA 50
11 51.5 ng/µl 1.029 0.574 1.79 1.87 DNA 50
12 44.4 ng/µl 0.889 0.488 1.82 2.14 DNA 50
13 51.9 ng/µl 1.039 0.573 1.81 1.93 DNA 50
14 54.9 ng/µl 1.098 0.6 1.83 2.19 DNA 50
15 63.8 ng/µl 1.277 0.703 1.82 2.21 DNA 50
16 55.1 ng/µl 1.102 0.594 1.86 2.27 DNA 50
17 64.6 ng/µl 1.292 0.7 1.85 2.25 DNA 50
18 68.3 ng/µl 1.366 0.741 1.84 2.19 DNA 50
19 79.1 ng/µl 1.583 0.867 1.83 2.18 DNA 50
20 58.6 ng/µl 1.173 0.653 1.8 2.14 DNA 50
21 45.1 ng/µl 0.902 0.498 1.81 1.84 DNA 50
22 66.2 ng/µl 1.323 0.734 1.8 2.2 DNA 50
23 70 ng/µl 1.4 0.755 1.86 2.19 DNA 50
24 45.3 ng/µl 0.906 0.499 1.82 2.16 DNA 50
25 44.3 ng/µl 0.887 0.487 1.82 2.21 DNA 50
26 66.2 ng/µl 1.323 0.722 1.83 2.14 DNA 50
27 49.8 ng/µl 0.995 0.545 1.83 2.16 DNA 50
28 51.7 ng/µl 1.034 0.567 1.82 2.05 DNA 50
29 45.2 ng/µl 0.905 0.506 1.79 1.96 DNA 50
30 76.8 ng/µl 1.535 0.837 1.83 2.11 DNA 50
31 48.4 ng/µl 0.968 0.544 1.78 2.12 DNA 50
32 37.3 ng/µl 0.746 0.419 1.78 1.96 DNA 50
33 28.7 ng/µl 0.574 0.329 1.75 1.72 DNA 50
34 38.5 ng/µl 0.771 0.428 1.8 1.93 DNA 50
35 39.5 ng/µl 0.789 0.446 1.77 1.85 DNA 50
36 44.2 ng/µl 0.883 0.479 1.85 2.13 DNA 50
37 42.6 ng/µl 0.853 0.471 1.81 2.07 DNA 50
38 32.3 ng/µl 0.646 0.357 1.81 1.92 DNA 50
39 49.1 ng/µl 0.982 0.546 1.8 1.99 DNA 50
40 43.7 ng/µl 0.875 0.483 1.81 1.98 DNA 50
41 48.6 ng/µl 0.971 0.548 1.77 2.08 DNA 50
42 46.2 ng/µl 0.925 0.502 1.84 2.15 DNA 50
43 43.5 ng/µl 0.87 0.49 1.78 1.96 DNA 50
44 46.7 ng/µl 0.934 0.516 1.81 2.12 DNA 50
45 55.1 ng/µl 1.103 0.591 1.87 1.98 DNA 50
46 52.5 ng/µl 1.05 0.584 1.8 2.12 DNA 50
47 37.6 ng/µl 0.751 0.42 1.79 2.02 DNA 50
48 50.1 ng/µl 1.002 0.548 1.83 2.07 DNA 50
49 59.7 ng/µl 1.194 0.652 1.83 2.11 DNA 50
50 33.8 ng/µl 0.676 0.385 1.75 1.97 DNA 50
51 42.4 ng/µl 0.848 0.481 1.76 1.93 DNA 50
52 66 ng/µl 1.321 0.729 1.81 2.16 DNA 50
53 81.2 ng/µl 1.625 0.895 1.81 2.06 DNA 50
54 42.1 ng/µl 0.842 0.476 1.77 2.05 DNA 50
55 39 ng/µl 0.78 0.458 1.7 1.8 DNA 50
56 28.9 ng/µl 0.579 0.347 1.67 1.91 DNA 50
57 51.6 ng/µl 1.031 0.577 1.79 2.12 DNA 50
58 52.3 ng/µl 1.045 0.602 1.74 2.12 DNA 50
59 57.5 ng/µl 1.15 0.639 1.8 2.1 DNA 50
60 57.2 ng/µl 1.144 0.644 1.78 2.06 DNA 50
61 49.4 ng/µl 0.989 0.577 1.71 2.03 DNA 50
62 59.8 ng/µl 1.196 0.674 1.78 1.91 DNA 50
63 42.7 ng/µl 0.855 0.491 1.74 1.99 DNA 50
64 45.5 ng/µl 0.91 0.517 1.76 1.8 DNA 50
65 43.8 ng/µl 0.877 0.49 1.79 1.91 DNA 50
66 37.4 ng/µl 0.748 0.419 1.79 2.07 DNA 50
67 37.9 ng/µl 0.757 0.437 1.74 1.86 DNA 50
68 31 ng/µl 0.619 0.368 1.68 1.8 DNA 50
69 39.4 ng/µl 0.789 0.445 1.77 2.05 DNA 50
70 44.8 ng/µl 0.895 0.521 1.72 1.63 DNA 50
71 37.8 ng/µl 0.755 0.444 1.7 1.86 DNA 50
72 41.8 ng/µl 0.836 0.481 1.74 2.03 DNA 50
73 64.2 ng/µl 1.284 0.72 1.78 2.06 DNA 50
74 40.1 ng/µl 0.803 0.452 1.78 1.82 DNA 50
75 33.6 ng/µl 0.673 0.382 1.76 2.06 DNA 50
76 26.5 ng/µl 0.531 0.31 1.71 2 DNA 50
A 51.5 ng/µl 1.029 0.571 1.8 2.17 DNA 50
B 51.6 ng/µl 1.033 0.575 1.8 1.86 DNA 50
C 79.3 ng/µl 1.587 0.889 1.79 2.22 DNA 50
D 55.2 ng/µl 1.105 0.626 1.77 2.01 DNA 50
E 49 ng/µl 0.98 0.561 1.75 2.05 DNA 50
F 36.7 ng/µl 0.735 0.438 1.68 1.83 DNA 50
G 51.5 ng/µl 1.029 0.59 1.74 2.04 DNA 50
H 48.7 ng/µl 0.974 0.56 1.74 1.88 DNA 50
I 61.2 ng/µl 1.224 0.695 1.76 2.05 DNA 50
CCA3 72.9 ng/µl 1.458 0.807 1.81 2.11 DNA 50
A. ricciae 105.2 ng/µl 2.104 1.147 1.84 2.21 DNA 50