User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/31

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Nuclear Gene Adenita Culture 454 Amplification

Culture 53, 55, 56, 58-66


  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 Chelex 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 53 Chelex B5 41 63 Chelex B3
2 53 Chelex C5 42 63 Chelex C3
3 53 Chelex D5 43 63 Chelex D3
4 53 Chelex E5 44 63 Chelex E3
5 53 Chelex F5 45 63 Chelex F3
6 55 Chelex B7 46 64 Chelex B4
7 55 Chelex C7 47 64 Chelex C4
8 55 Chelex D7 48 64 Chelex D4
9 55 Chelex E7 49 64 Chelex E4
10 55 Chelex F7 50 64 Chelex F4
11 56 Chelex B8 51 65 Chelex B5
12 56 Chelex C8 52 65 Chelex C5
13 56 Chelex D8 53 65 Chelex D5
14 56 Chelex E8 54 65 Chelex E5
15 56 Chelex F8 55 65 Chelex F5
16 58 Chelex B10 56 66 Chelex B6
17 58 Chelex C10 57 66 Chelex C6
18 58 Chelex D10 58 66 Chelex D6
19 58 Chelex E10 59 66 Chelex E6
20 58 Chelex F10 60 66 Chelex F6
21 59 Chelex B11 61 Extraction Control B3
22 59 Chelex C11 62 Extraction Control C3
23 59 Chelex D11 63 Extraction Control D3
24 59 Chelex E11 64 Extraction Control E3
25 59 Chelex F11 65 Extraction Control F3
26 60 Chelex B12 66 Neg Control B3
27 60 Chelex C12 67 Neg Control C3
28 60 Chelex D12 68 Neg Control D3
29 60 Chelex E12 69 Neg Control E3
30 60 Chelex F12 70 Neg Control F3
31 61 Chelex B1 71 Pos Control B3
32 61 Chelex C1 72 Pos Control C3
33 61 Chelex D1 73 Pos Control D3
34 61 Chelex E1 74 Pos Control E3
35 61 Chelex F1 75 Pos Control F3
36 62 Chelex B2
37 62 Chelex C2
38 62 Chelex D2
39 62 Chelex E2
40 62 Chelex F2
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul

31May10A.jpg

Nuclear Gene Adenita Culture 454 Amplification

Culture 67, 68, 71-76, 11, 21, 27, 28


  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 Chelex 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 67 Chelex B7 41 11 Chelex B11
2 67 Chelex C7 42 11 Chelex C11
3 67 Chelex D7 43 11 Chelex D11
4 67 Chelex E7 44 11 Chelex E11
5 67 Chelex F7 45 11 Chelex F11
6 68 Chelex B8 46 21 Chelex B9
7 68 Chelex C8 47 21 Chelex C9
8 68 Chelex D8 48 21 Chelex D9
9 68 Chelex E8 49 21 Chelex E9
10 68 Chelex F8 50 21 Chelex F9
11 71 Chelex B11 51 27 Chelex B3
12 71 Chelex C11 52 27 Chelex C3
13 71 Chelex D11 53 27 Chelex D3
14 71 Chelex E11 54 27 Chelex E3
15 71 Chelex F11 55 27 Chelex F3
16 72 Chelex B12 56 28 Chelex B4
17 72 Chelex C12 57 28 Chelex C4
18 72 Chelex D12 58 28 Chelex D4
19 72 Chelex E12 59 28 Chelex E4
20 72 Chelex F12 60 28 Chelex F4
21 73 Chelex B1 61 Extraction Control B7
22 73 Chelex C1 62 Extraction Control C7
23 73 Chelex D1 63 Extraction Control D7
24 73 Chelex E1 64 Extraction Control E7
25 73 Chelex F1 65 Extraction Control F7
26 74 Chelex B2 66 Neg Control B7
27 74 Chelex C2 67 Neg Control C7
28 74 Chelex D2 68 Neg Control D7
29 74 Chelex E2 69 Neg Control E7
30 74 Chelex F2 70 Neg Control F7
31 75 Chelex B3 71 Pos Control B7
32 75 Chelex C3 72 Pos Control C7
33 75 Chelex D3 73 Pos Control D7
34 75 Chelex E3 74 Pos Control E7
35 75 Chelex F3 75 Pos Control F7
36 76 Chelex B4
37 76 Chelex C4
38 76 Chelex D4
39 76 Chelex E4
40 76 Chelex F4
*Positive Control = AV 20ng
** Extraction Control = AV Chelex


Load 2.0ul

31May10B.jpg