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

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Contents

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 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
110 ChelexB103148 ChelexB12
210 ChelexC103248 ChelexC12
310 ChelexD103348 ChelexD12
410 ChelexE103448 ChelexE12
510 ChelexF103548 ChelexF12
643 ChelexB7361 ChelexB1
743 ChelexC7371 ChelexC1
843 ChelexD7381 ChelexD1
943 ChelexE7391 ChelexE1
1043 ChelexF7401 ChelexF1
1144 ChelexB841Extraction ControlB10
1244 ChelexC842Extraction ControlC10
1344 ChelexD843Extraction ControlD10
1444 ChelexE844Extraction ControlE10
1544 ChelexF845Extraction ControlF10
1645 ChelexB946Neg ControlB10
1745 ChelexC947Neg ControlC10
1845 ChelexD948Neg ControlD10
1945 ChelexE949Neg ControlE10
2045 ChelexF950Neg ControlF10
2146 ChelexB1051Pos ControlB10
2246 ChelexC1052Pos ControlC10
2346 ChelexD1053Pos ControlD10
2446 ChelexE1054Pos ControlE10
2546 ChelexF1055Pos ControlF10
2647 ChelexB11
2747 ChelexC11
2847 ChelexD11
2947 ChelexE11
3047 ChelexF11
*Positive Control = AV 20ng
** Extraction Control = AV Chelex


Load 2.0ul

Image: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 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
149 ChelexB121Extraction ControlB3
249 ChelexC122Extraction ControlC3
349 ChelexD123Extraction ControlD3
449 ChelexE124Extraction ControlE3
549 ChelexF125Extraction ControlF3
651 ChelexB326Neg ControlB3
751 ChelexC327Neg ControlC3
851 ChelexD328Neg ControlD3
951 ChelexE329Neg ControlE3
1051 ChelexF330Neg ControlF3
1152 ChelexB431Pos ControlB3
1252 ChelexC432Pos ControlC3
1352 ChelexD433Pos ControlD3
1452 ChelexE434Pos ControlE3
1552 ChelexF435Pos ControlF3
1653 ChelexB5
1753 ChelexC5
1853 ChelexD5
1953 ChelexE5
2053 ChelexF5
*Positive Control = AV 20ng
** Extraction Control = AV Chelex

Load 2.0ul

Image:28May10B.jpg

454 adapter preparation

Adapter Conc. 1x ' Temp time
Adapter_A-Biotin95 C2 min
Primer_A(454)-5\'Biotin100 uM50 ul
Primer_A(454)RevCon-5\'P100 uM50 ulto 30 Cfor 40 min
Adapter_B20 C1 min
Primer_B(454)100 uM50 ul
Primer_B(454)RevCon-5\'P100 uM50 ul
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