User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/07

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Nuclear Gene Adenita Culture 454 Amplification

Adenita Cultures 11, 27-29, 31, 32, 39-41, 43, 50, 51, 54-56, H 

  1. Adenita cultures 5-15 individuals.
  2. Spin for 1 min at 12-14,000 rpm.  Remove supernatant leaving 20-30ul, careful not to disturb pellet.
  3. Add 100ul 20% chelex (Or add chelex until ~1/2 tube full of beads).  Vortex, then quick spin.
  4. Boil for 10 minutes.
  5. Before use, vortex, then spin at 13000 rpm for 3 minutes.  Use supernatant right above beads.


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 11 Chelex B11 51 50 Chelex B2
2 11 Chelex C11 52 50 Chelex C2
3 11 Chelex D11 53 50 Chelex D2
4 11 Chelex E11 54 50 Chelex E2
5 11 Chelex F11 55 50 Chelex F2
6 27 Chelex B3 56 51 Chelex B3
7 27 Chelex C3 57 51 Chelex C3
8 27 Chelex D3 58 51 Chelex D3
9 27 Chelex E3 59 51 Chelex E3
10 27 Chelex F3 60 51 Chelex F3
11 28 Chelex B4 61 54 Chelex B6
12 28 Chelex C4 62 54 Chelex C6
13 28 Chelex D4 63 54 Chelex D6
14 28 Chelex E4 64 54 Chelex E6
15 28 Chelex F4 65 54 Chelex F6
16 29 Chelex B5 66 55 Chelex B7
17 29 Chelex C5 67 55 Chelex C7
18 29 Chelex D5 68 55 Chelex D7
19 29 Chelex E5 69 55 Chelex E7
20 29 Chelex F5 70 55 Chelex F7
21 31 Chelex B7 71 56 Chelex B8
22 31 Chelex C7 72 56 Chelex C8
23 31 Chelex D7 73 56 Chelex D8
24 31 Chelex E7 74 56 Chelex E8
25 31 Chelex F7 75 56 Chelex F8
26 32 Chelex B8 76 H Chelex B12
27 32 Chelex C8 77 H Chelex C12
28 32 Chelex D8 78 H Chelex D12
29 32 Chelex E8 79 H Chelex E12
30 32 Chelex F8 80 H Chelex F12
31 39 Chelex B3 81 Extraction Control B12
32 39 Chelex C3 82 Extraction Control C12
33 39 Chelex D3 83 Extraction Control D12
34 39 Chelex E3 84 Extraction Control E12
35 39 Chelex F3 85 Extraction Control F12
36 40 Chelex B4 86 Neg Control B12
37 40 Chelex C4 87 Neg Control C12
38 40 Chelex D4 88 Neg Control D12
39 40 Chelex E4 89 Neg Control E12
40 40 Chelex F4 90 Neg Control F12
41 41 Chelex B5 91 Pos Control B12
42 41 Chelex C5 92 Pos Control C12
43 41 Chelex D5 93 Pos Control D12
44 41 Chelex E5 94 Pos Control E12
45 41 Chelex F5 95 Pos Control F12
46 43 Chelex B7
47 43 Chelex C7
48 43 Chelex D7
49 43 Chelex E7
50 43 Chelex F7

Load 2.0ul Sample

- rerun negative control on gel

- weak band in negative control

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