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

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Nuclear Gene Adenita 454 Amplification

Test AV10027 Primer set on Instagene Extractions

EX Takara RXN Mixture conc 1X 14
Milli Q water 4.15 58.1
Takara EX Buffer 10X 1 14
dNTP 2.5uM 0.8 11.2
Primer Mix AV10027 5uM 1 14
EX Taq 5U 0.05 0.7
Template Instagene 3
98
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Extraction
1 1 Instagene
2 2 Instagene
3 10 Instagene old
4 11 Instagene old
5 10 Instagene
6 11 Instagene
7 33 Instagene
8 34 Instagene
9 35 Instagene
10 40 Instagene
11 41 Instagene
12 42 Instagene
13 Negative Control
14 Positive Control
*Positive Control = AV 20ng

Loaded 2.0ul

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 water 4.15 24.9
Takara EX Buffer 10X 1 6
dNTP 2.5uM 0.8 4.8
Primer Mix AV10027 5uM 1 6
EX Taq 5U 0.05 0.3
Template Chelex 3
42
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Extraction
1 1 Chelex Short Chelex
2 10 Chelex Short Chelex
3 AV Chelex Short Chelex
4 AV Chelex Short Chelex
5 Negative Control
6 Positive Control
*Positive Control = AV 20ng

Load 2.0ul

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 water 4.15 24.9
Takara EX Buffer 10X 1 6
dNTP 2.5uM 0.8 4.8
Primer Mix AV10027 5uM 1 6
EX Taq 5U 0.05 0.3
Template Chelex 3
42
7.0ul / RXN add 3.0ul Sample
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Extraction
1 1 Chelex Long Chelex
2 10 Chelex Long Chelex
3 AV Chelex Long Chelex
4 AV Chelex Long Chelex
5 Negative Control
6 Positive Control
*Positive Control = AV 20ng


Load 2.0ul

27May10C.jpg