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

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Contents

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 water4.1520.75
Takara EX Buffer10X15
dNTP2.5uM0.84
Primer Mix20uM1
EX Taq5U0.050.25
TemplateInstagene315
45
9.0ul / RXN add 1.0ul Primer
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplatePrimerAMP LaneTemplatePrimer
157 InstageneB931F InstageneB10
257 InstageneC932F InstageneC10
357 InstageneD933F InstageneD10
457 InstageneE934F InstageneE10
557 InstageneF935F InstageneF10
6A InstageneB536G InstageneB11
7A InstageneC537G InstageneC11
8A InstageneD538G InstageneD11
9A InstageneE539G InstageneE11
10A InstageneF540G InstageneF11
11B InstageneB641H InstageneB12
12B InstageneC642H InstageneC12
13B InstageneD643H InstageneD12
14B InstageneE644H InstageneE12
15B InstageneF645H InstageneF12
16C InstageneB746I InstageneB1
17C InstageneC747I InstageneC1
18C InstageneD748I InstageneD1
19C InstageneE749I InstageneE1
20C InstageneF750I InstageneF1
21D InstageneB851Neg ControlB9
22D InstageneC852Neg ControlC9
23D InstageneD853Neg ControlD9
24D InstageneE854Neg ControlE9
25D InstageneF855Neg ControlF9
26E InstageneB956Pos ControlB9
27E InstageneC957Pos ControlC9
28E InstageneD958Pos ControlD9
29E InstageneE959Pos ControlE9
30E InstageneF960Pos ControlF9
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul


Image: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 water4.1520.75
Takara EX Buffer10X15
dNTP2.5uM0.84
Primer Mix20uM1
EX Taq5U0.050.25
TemplateChelex315
45
9.0ul / RXN add 1.0ul Primer
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplatePrimerAMP LaneTemplatePrimer
129 ChelexB54639 ChelexB3
229 ChelexC54739 ChelexC3
329 ChelexD54839 ChelexD3
429 ChelexE54939 ChelexE3
529 ChelexF55039 ChelexF3
631 ChelexB75140 ChelexB4
731 ChelexC75240 ChelexC4
831 ChelexD75340 ChelexD4
931 ChelexE75440 ChelexE4
1031 ChelexF75540 ChelexF4
1132 ChelexB85641 ChelexB5
1232 ChelexC85741 ChelexC5
1332 ChelexD85841 ChelexD5
1432 ChelexE85941 ChelexE5
1532 ChelexF86041 ChelexF5
1633 ChelexB96142 ChelexB6
1733 ChelexC96242 ChelexC6
1833 ChelexD96342 ChelexD6
1933 ChelexE96442 ChelexE6
2033 ChelexF96542 ChelexF6
2134 ChelexB106643 ChelexB7
2234 ChelexC106743 ChelexC7
2334 ChelexD106843 ChelexD7
2434 ChelexE106943 ChelexE7
2534 ChelexF107043 ChelexF7
2635 ChelexB117150 ChelexB2
2735 ChelexC117250 ChelexC2
2835 ChelexD117350 ChelexD2
2935 ChelexE117450 ChelexE2
3035 ChelexF117550 ChelexF2
3136 ChelexB1276Extraction ControlB5
3236 ChelexC1277Extraction ControlC5
3336 ChelexD1278Extraction ControlD5
3436 ChelexE1279Extraction ControlE5
3536 ChelexF1280Extraction ControlF5
3637 ChelexB181Neg ControlB5
3737 ChelexC182Neg ControlC5
3837 ChelexD183Neg ControlD5
3937 ChelexE184Neg ControlE5
4037 ChelexF185Neg ControlF5
4138 ChelexB286Pos ControlB5
4238 ChelexC287Pos ControlC5
4338 ChelexD288Pos ControlD5
4438 ChelexE289Pos ControlE5
4538 ChelexF290Pos ControlF5
*Positive Control = AV 20ng
** Extraction Control = AV Chelex


Load 2.0ul


Image:01June10B.jpg

LCO/HCO Amplification

EX Takara RXN Mixture conc 1X 6 '
Milli Q water10.361.8
Takara EX Buffer10X212
dNTP2.5uM1.69.6
Primer Mix20uM212
EX Taq5U0.10.6
TemplateInstagene4
96
16.0ul / RXN add 4.0ul Template
PCR cycle
94C3 min
94C10 sec
45-68C3 min
RAMP 0.5C/sec
x35
68C5 min
AMP Lane Template ' Sample Number
1E. WindsorB28
2Ithaca 1A112
3Woods Hole 1C4G
4Neg Control
5AV Positive Control


Load 2.0ul

Image: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

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

>800bp

Image: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)0.5MNEB 2log marker
TopTaq Buffer10x13.51<300 bp
dNTP mix10 mM each0.20.72300bp to 800bp
Primer mix (454Primer A and B)5 uM each13.53>800 bp
TopTaq5 U/ul0.050.175
Milli Q7.2525.375
10 ul
PCR cycle
94C3 min
98C10 sec
60C1 min
x35
72C5 min

Image:0099.jpg

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