User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/27

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Contents

Nuclear Gene Adenita 454 Amplification

Test AV10027 Primer set on Instagene Extractions

EX Takara RXN Mixture conc 1X 14
Milli Q water4.1558.1
Takara EX Buffer10X114
dNTP2.5uM0.811.2
Primer Mix AV10027 5uM114
EX Taq5U0.050.7
TemplateInstagene3
98
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplateExtraction
11 Instagene
22 Instagene
310 Instagene old
411 Instagene old
510 Instagene
611 Instagene
733 Instagene
834 Instagene
935 Instagene
1040 Instagene
1141 Instagene
1242 Instagene
13Negative Control
14Positive Control
*Positive Control = AV 20ng

Loaded 2.0ul

Image:27May10A.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 200ul 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 6
Milli Q water4.1524.9
Takara EX Buffer10X16
dNTP2.5uM0.84.8
Primer Mix AV10027 5uM16
EX Taq5U0.050.3
TemplateChelex3
42
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplateExtraction
11 ChelexShort Chelex
210 ChelexShort Chelex
3AV ChelexShort Chelex
4AV ChelexShort Chelex
5Negative Control
6Positive Control
*Positive Control = AV 20ng

Load 2.0ul

Image:27May10B.jpg

Nuclear Gene Adenita 454 Test Chelex Extraction

If the short version doesn’t work, try the long version:

  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 200ul 20 % chelex.  (Or add chelex until ~1/2 tube full of beads).  Vortex, then quick spin. 
  4. Incubate at 55 degrees for 1 hour
  5. Vortex for 10 seconds.
  6. Boil for 10 minutes.  
  7. Before use, spin at 13000 rpm for 3 minutes.
EX Takara RXN Mixture conc 1X 6
Milli Q water4.1524.9
Takara EX Buffer10X16
dNTP2.5uM0.84.8
Primer Mix AV10027 5uM16
EX Taq5U0.050.3
TemplateChelex3
42
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C10 sec
60C1 min
x35
AMP LaneTemplateExtraction
11 ChelexLong Chelex
210 ChelexLong Chelex
3AV ChelexLong Chelex
4AV ChelexLong Chelex
5Negative Control
6Positive Control
*Positive Control = AV 20ng


Load 2.0ul

Image:27May10C.jpg

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