User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/01

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Nuclear Gene Adenita Culture 454 Amplification

Re-Amp Instagene Culture 57, A, B, C, D, E, F, G, H, 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 57 Instagene B9 31 F Instagene B10
2 57 Instagene C9 32 F Instagene C10
3 57 Instagene D9 33 F Instagene D10
4 57 Instagene E9 34 F Instagene E10
5 57 Instagene F9 35 F Instagene F10
6 A Instagene B5 36 G Instagene B11
7 A Instagene C5 37 G Instagene C11
8 A Instagene D5 38 G Instagene D11
9 A Instagene E5 39 G Instagene E11
10 A Instagene F5 40 G Instagene F11
11 B Instagene B6 41 H Instagene B12
12 B Instagene C6 42 H Instagene C12
13 B Instagene D6 43 H Instagene D12
14 B Instagene E6 44 H Instagene E12
15 B Instagene F6 45 H Instagene F12
16 C Instagene B7 46 I Instagene B1
17 C Instagene C7 47 I Instagene C1
18 C Instagene D7 48 I Instagene D1
19 C Instagene E7 49 I Instagene E1
20 C Instagene F7 50 I Instagene F1
21 D Instagene B8 51 Neg Control B9
22 D Instagene C8 52 Neg Control C9
23 D Instagene D8 53 Neg Control D9
24 D Instagene E8 54 Neg Control E9
25 D Instagene F8 55 Neg Control F9
26 E Instagene B9 56 Pos Control B9
27 E Instagene C9 57 Pos Control C9
28 E Instagene D9 58 Pos Control D9
29 E Instagene E9 59 Pos Control E9
30 E Instagene F9 60 Pos Control F9
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul


01June10A.jpg


Nuclear Gene Adenita Culture 454 Amplification

Culture 29, 31-43, 50


  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 29 Chelex B5 46 39 Chelex B3
2 29 Chelex C5 47 39 Chelex C3
3 29 Chelex D5 48 39 Chelex D3
4 29 Chelex E5 49 39 Chelex E3
5 29 Chelex F5 50 39 Chelex F3
6 31 Chelex B7 51 40 Chelex B4
7 31 Chelex C7 52 40 Chelex C4
8 31 Chelex D7 53 40 Chelex D4
9 31 Chelex E7 54 40 Chelex E4
10 31 Chelex F7 55 40 Chelex F4
11 32 Chelex B8 56 41 Chelex B5
12 32 Chelex C8 57 41 Chelex C5
13 32 Chelex D8 58 41 Chelex D5
14 32 Chelex E8 59 41 Chelex E5
15 32 Chelex F8 60 41 Chelex F5
16 33 Chelex B9 61 42 Chelex B6
17 33 Chelex C9 62 42 Chelex C6
18 33 Chelex D9 63 42 Chelex D6
19 33 Chelex E9 64 42 Chelex E6
20 33 Chelex F9 65 42 Chelex F6
21 34 Chelex B10 66 43 Chelex B7
22 34 Chelex C10 67 43 Chelex C7
23 34 Chelex D10 68 43 Chelex D7
24 34 Chelex E10 69 43 Chelex E7
25 34 Chelex F10 70 43 Chelex F7
26 35 Chelex B11 71 50 Chelex B2
27 35 Chelex C11 72 50 Chelex C2
28 35 Chelex D11 73 50 Chelex D2
29 35 Chelex E11 74 50 Chelex E2
30 35 Chelex F11 75 50 Chelex F2
31 36 Chelex B12 76 Extraction Control B5
32 36 Chelex C12 77 Extraction Control C5
33 36 Chelex D12 78 Extraction Control D5
34 36 Chelex E12 79 Extraction Control E5
35 36 Chelex F12 80 Extraction Control F5
36 37 Chelex B1 81 Neg Control B5
37 37 Chelex C1 82 Neg Control C5
38 37 Chelex D1 83 Neg Control D5
39 37 Chelex E1 84 Neg Control E5
40 37 Chelex F1 85 Neg Control F5
41 38 Chelex B2 86 Pos Control B5
42 38 Chelex C2 87 Pos Control C5
43 38 Chelex D2 88 Pos Control D5
44 38 Chelex E2 89 Pos Control E5
45 38 Chelex F2 90 Pos Control F5
*Positive Control = AV 20ng
** Extraction Control = AV Chelex


Load 2.0ul


01June10B.jpg

LCO/HCO Amplification

EX Takara RXN Mixture conc 1X 6 '
Milli Q water 10.3 61.8
Takara EX Buffer 10X 2 12
dNTP 2.5uM 1.6 9.6
Primer Mix 20uM 2 12
EX Taq 5U 0.1 0.6
Template Instagene 4
96
16.0ul / RXN add 4.0ul Template
PCR cycle
94C 3 min
94C 10 sec
45-68C 3 min
RAMP 0.5C/sec
x35
68C 5 min
AMP Lane Template ' Sample Number
1 E. Windsor B2 8
2 Ithaca 1 A1 12
3 Woods Hole 1 C4 G
4 Neg Control
5 AV Positive Control


Load 2.0ul

01June10C.jpg

454 library preparation (Alien Genes Mix)

Purification of adapter ligated sample by AMPure beads (modified protocol by Lennon et al. 2010)

  1. Add 25ul of AMPure beads (x0.5) to 50ul of sample.
  2. Pipette 10 times.
  3. Incubate for 5 min at r.t.
  4. Incubate the mixture on the magnet plate for 5 min at r.t.
  5. Transfer the supernatant to a new 0.2ml tube.
  6. Add 200 of 70% EtOH to the beads (for <300bp).
  7. Add 53.2ul of AMPure (x0.7) beads to the supernatant.
  8. Repeat steps 2 to 6 (for 300 to 800bp).
  9. Add 95.8ul of AMPure (x1.8) beads to the supernatant.
  10. Repeat steps 2 to 4.
  11. Discard the supernatant.
  12. Add 200 of 70% EtOH to the beads (for >800bp).
  13. Repeat steps 11 and 12.
  14. Discard the supernatant.
  15. Off the magnet plate, add 40 ul of buffer EB (QIAGEN) to the beads.
  16. Pipette 10 times.
  17. Incubate on the magnet plate for 1 min.
  18. Transfer the supernatant to a new 1.5ml tube.

Sample of 300 to 800bp was run out for the next step.

<300bp

2100 expert High Sensitivity DNA Assay DE54704331 2010-06-09 16-29-15 page8.jpg

>800bp

2100 expert High Sensitivity DNA Assay DE54704331 2010-06-09 16-29-15 page9.jpg

Confirmation of the library by PCR

Reaction mixture conc. 1x x3.5 ' lane '
Temp (<300bp, 300to800bp, >800bp) 0.5 M NEB 2log marker
TopTaq Buffer 10x 1 3.5 1 <300 bp
dNTP mix 10 mM each 0.2 0.7 2 300bp to 800bp
Primer mix (454Primer A and B) 5 uM each 1 3.5 3 >800 bp
TopTaq 5 U/ul 0.05 0.175
Milli Q 7.25 25.375
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 1 min
x35
72C 5 min

0099.jpg