User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/07

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Contents

Nuclear Gene Adenita Culture 454 Amplification

Adenita Cultures 11, 27-29, 31, 32, 39-41, 43, 50, 51, 54-56, H

  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
TemplateInstagene315
45
9.0ul / RXN add 1.0ul Primer
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplatePrimerAMP LaneTemplatePrimer
111 ChelexB115150 ChelexB2
211 ChelexC115250 ChelexC2
311 ChelexD115350 ChelexD2
411 ChelexE115450 ChelexE2
511 ChelexF115550 ChelexF2
627 ChelexB35651 ChelexB3
727 ChelexC35751 ChelexC3
827 ChelexD35851 ChelexD3
927 ChelexE35951 ChelexE3
1027 ChelexF36051 ChelexF3
1128 ChelexB46154 ChelexB6
1228 ChelexC46254 ChelexC6
1328 ChelexD46354 ChelexD6
1428 ChelexE46454 ChelexE6
1528 ChelexF46554 ChelexF6
1629 ChelexB56655 ChelexB7
1729 ChelexC56755 ChelexC7
1829 ChelexD56855 ChelexD7
1929 ChelexE56955 ChelexE7
2029 ChelexF57055 ChelexF7
2131 ChelexB77156 ChelexB8
2231 ChelexC77256 ChelexC8
2331 ChelexD77356 ChelexD8
2431 ChelexE77456 ChelexE8
2531 ChelexF77556 ChelexF8
2632 ChelexB876H ChelexB12
2732 ChelexC877H ChelexC12
2832 ChelexD878H ChelexD12
2932 ChelexE879H ChelexE12
3032 ChelexF880H ChelexF12
3139 ChelexB381Extraction ControlB12
3239 ChelexC382Extraction ControlC12
3339 ChelexD383Extraction ControlD12
3439 ChelexE384Extraction ControlE12
3539 ChelexF385Extraction ControlF12
3640 ChelexB486Neg ControlB12
3740 ChelexC487Neg ControlC12
3840 ChelexD488Neg ControlD12
3940 ChelexE489Neg ControlE12
4040 ChelexF490Neg ControlF12
4141 ChelexB591Pos ControlB12
4241 ChelexC592Pos ControlC12
4341 ChelexD593Pos ControlD12
4441 ChelexE594Pos ControlE12
4541 ChelexF595Pos ControlF12
4643 ChelexB7
4743 ChelexC7
4843 ChelexD7
4943 ChelexE7
5043 ChelexF7

Load 2.0ul Sample

Image:07June10.jpg

- rerun negative control on gel

Image:07June10 negative control reload.jpg

- weak band in negative control

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