User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/28

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Nuclear Gene Adenita 454 Test Chelex Extraction

  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 10 Chelex B10 31 48 Chelex B12
2 10 Chelex C10 32 48 Chelex C12
3 10 Chelex D10 33 48 Chelex D12
4 10 Chelex E10 34 48 Chelex E12
5 10 Chelex F10 35 48 Chelex F12
6 43 Chelex B7 36 1 Chelex B1
7 43 Chelex C7 37 1 Chelex C1
8 43 Chelex D7 38 1 Chelex D1
9 43 Chelex E7 39 1 Chelex E1
10 43 Chelex F7 40 1 Chelex F1
11 44 Chelex B8 41 Extraction Control B10
12 44 Chelex C8 42 Extraction Control C10
13 44 Chelex D8 43 Extraction Control D10
14 44 Chelex E8 44 Extraction Control E10
15 44 Chelex F8 45 Extraction Control F10
16 45 Chelex B9 46 Neg Control B10
17 45 Chelex C9 47 Neg Control C10
18 45 Chelex D9 48 Neg Control D10
19 45 Chelex E9 49 Neg Control E10
20 45 Chelex F9 50 Neg Control F10
21 46 Chelex B10 51 Pos Control B10
22 46 Chelex C10 52 Pos Control C10
23 46 Chelex D10 53 Pos Control D10
24 46 Chelex E10 54 Pos Control E10
25 46 Chelex F10 55 Pos Control F10
26 47 Chelex B11
27 47 Chelex C11
28 47 Chelex D11
29 47 Chelex E11
30 47 Chelex F11
*Positive Control = AV 20ng
** Extraction Control = AV Chelex


Load 2.0ul

28May10A.jpg

Nuclear Gene Adenita 454 Test Chelex Extraction

  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 49 Chelex B1 21 Extraction Control B3
2 49 Chelex C1 22 Extraction Control C3
3 49 Chelex D1 23 Extraction Control D3
4 49 Chelex E1 24 Extraction Control E3
5 49 Chelex F1 25 Extraction Control F3
6 51 Chelex B3 26 Neg Control B3
7 51 Chelex C3 27 Neg Control C3
8 51 Chelex D3 28 Neg Control D3
9 51 Chelex E3 29 Neg Control E3
10 51 Chelex F3 30 Neg Control F3
11 52 Chelex B4 31 Pos Control B3
12 52 Chelex C4 32 Pos Control C3
13 52 Chelex D4 33 Pos Control D3
14 52 Chelex E4 34 Pos Control E3
15 52 Chelex F4 35 Pos Control F3
16 53 Chelex B5
17 53 Chelex C5
18 53 Chelex D5
19 53 Chelex E5
20 53 Chelex F5
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul

28May10B.jpg

454 adapter preparation

Adapter Conc. 1x ' Temp time
Adapter_A-Biotin 95 C 2 min
Primer_A(454)-5\'Biotin 100 uM 50 ul
Primer_A(454)RevCon-5\'P 100 uM 50 ul to 30 C for 40 min
Adapter_B 20 C 1 min
Primer_B(454) 100 uM 50 ul
Primer_B(454)RevCon-5\'P 100 uM 50 ul