User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/03

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Nuclear Gene Adenita 454 Amplification

Adenita Culture 54, 69, 70, A, B, E, F, G, CCA3

  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 54 Chelex B6 31 F Chelex B10
2 54 Chelex C6 32 F Chelex C10
3 54 Chelex D6 33 F Chelex D10
4 54 Chelex E6 34 F Chelex E10
5 54 Chelex F6 35 F Chelex F10
6 69 Chelex B9 36 G Chelex B11
7 69 Chelex C9 37 G Chelex C11
8 69 Chelex D9 38 G Chelex D11
9 69 Chelex E9 39 G Chelex E11
10 69 Chelex F9 40 G Chelex F11
11 70 Chelex B10 41 CCA3 Chelex B2
12 70 Chelex C10 42 CCA3 Chelex C2
13 70 Chelex D10 43 CCA3 Chelex D2
14 70 Chelex E10 44 CCA3 Chelex E2
15 70 Chelex F10 45 CCA3 Chelex F2
16 A Chelex B5 46 Extraction Control B6
17 A Chelex C5 47 Extraction Control C6
18 A Chelex D5 48 Extraction Control D6
19 A Chelex E5 49 Extraction Control E6
20 A Chelex F5 50 Extraction Control F6
21 B Chelex B6 51 Neg Control B6
22 B Chelex C6 52 Neg Control C6
23 B Chelex D6 53 Neg Control D6
24 B Chelex E6 54 Neg Control E6
25 B Chelex F6 55 Neg Control F6
26 E Chelex B9 56 Pos Control B6
27 E Chelex C9 57 Pos Control C6
28 E Chelex D9 58 Pos Control D6
29 E Chelex E9 59 Pos Control E6
30 E Chelex F9 60 Pos Control F6
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul

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