User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/16

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Contents

CCA3 Qiagen Extraction

~100-200 individuals Used 1mM NaCl to make rotifers curl, then moved to extraction tube

Followed standard Qiagen tissue extraction protocol

Nanodrop DNA Tube (1) 6.9 ng/ul Tube (2) 3.1 ng/ul

R Takara Test Nuclear Genes

R Takara RXN Mixture conc 1X 6.5 '
Milli Q water4.5529.575
Takara R Buffer10X16.5
MgCl225uM0.63.9
dNTP2.5uM0.85.2
Primer Mix5uM1
R Taq5U0.050.325
Templateinstagene213
58.59.0ul / RXN
A PCR cycle
95C3 min
95C5 sec
60C1 min
x35
AMP Lane Template Primer
171 InstageneA1
271 InstageneA2
371 InstageneA3
471 InstageneA4
571 InstageneA5
671 InstageneA6
772 InstageneA1
872 InstageneA2
972 InstageneA3
1072 InstageneA4
1172 InstageneA5
1272 InstageneA6
13AV InstageneA1
14AV InstageneA2
15AV InstageneA3
16AV InstageneA4
17AV InstageneA5
18AV InstageneA6
* AV Positive Control

Load 2.0ul sample

Image:16April10.tif

R Takara Test and HS Takara Test Nuclear Genes

R Takara RXN Mixture conc 1X 6.5 '
Milli Q water4.5529.575
Takara R Buffer10X16.5
MgCl225uM1.27.8
dNTP2.5uM0.85.2
Primer Mix5uM1
R Taq5U0.050.325
TemplateInstagene213
62.49.0ul / RXN
HS Takara RXN Mixtureconc1X6.5
Milli Q water4.126.65
Takara HS Buffer10X213
dNTP2.5uM0.85.2
Primer Mix5uM1
HS Taq5U0.10.65
TemplateInstagene213
58.59.0ul / RXN
A PCR cycle
95C3 min
95C5 sec
60C1 min
x35
*AV Positive Control
AMP Lane Template Primer
171 InstageneA1
271 InstageneA2
371 InstageneA3
471 InstageneA4
571 InstageneA5
671 InstageneA6
772 InstageneA1
872 InstageneA2
972 InstageneA3
1072 InstageneA4
1172 InstageneA5
1272 InstageneA6
13AV InstageneA1
14AV InstageneA2
15AV InstageneA3
16AV InstageneA4
17AV InstageneA5
18AV InstageneA6
1971 InstageneA1
2071 InstageneA2
2171 InstageneA3
2271 InstageneA4
2371 InstageneA5
2471 InstageneA6
2572 InstageneA1
2672 InstageneA2
2772 InstageneA3
2872 InstageneA4
2972 InstageneA5
3072 InstageneA6
31AV InstageneA1
32AV InstageneA2
33AV InstageneA3
34AV InstageneA4
35AV InstageneA5
36AV InstageneA6

Load 2.0ul sample

Image:16April10 B.tif

PCR test (TopTaq)

Reaction mixture conc. 1x x3.5 ' lane '
Temp (InstaGene)??21A.vaga
TopTaq Buffer10x13.52CC A3
dNTP mix10 mM each0.20.73Tsukuba D4 (negative control)
Primer mix (AV10027)5 uM each13.5
TopTaq5 U/ul0.050.175
Milli Q5.7520.125
10 ul
PCR cycle
94C3 min
98C10 sec
60C3 min
x35
72C5 min


load 2.5ul of sample

Image:0082.jpg

Alien Genes New Primer Screen

Reaction mixture conc. 1x 32.5 ' Lane Gene Lane Gene
Temp (Tsukuba D4, InstaGene)??2651AV1015817AV10084
TopTaq Buffer10x132.52AV1015318AV10082
dNTP mix10 mM each0.26.53AV1015119AV10075
Primer mix5 uM each14AV1015020AV10071
TopTaq5 U/ul0.051.6255AV1014021AV10045
Milli Q5.75186.8756AV1013422AV10043
10 ul7AV1013023AV10042
8AV1012324AV10041
PCR cycle9AV1012125AV10034
94C3 min10AV1010426AV10028
11AV1010927AV10027
94C10 sec12AV1009928AV10025
60C3 min13AV1009829AV10016
x3514AV1009430AV10011
15AV1009231AV10007
72C5 min16AV1008932AV10002

load 2.5ul of sample

Image:0084.jpg

Gene-rich region of CCA3 -- take 2

  • this time going to try the same thing but with higher DNA concentration

PCR conditions

Reaction mixture conc. 1x 12.5
water5.3566.875
LA buffer10x112.5
dNTP mix2.5 mM each1.620
Primer mix5 uM each1
TakaraLA5U/ul0.050.625
template3 ng/ul112.5
9
  • Primers:
1. Avhsp-1_2-1R7. Avhsp-3_1-2F
2. Avhsp-1_3-2F8. Avhsp-4_1-2F
3. Avhsp-1_6-1F9. Avhsp-4_3-1F
4. Avhsp-1_7-1F10. Avhis-2_2-2F
5. Avhsp-1_7-2F11. Avhsp-4_2-1F
6. Avhsp-3_1-1R12. Avhsp-4_2-2F
  • 94C 1 min, (98C, 10 sec;65C 10 min)x30, 72C 5 min

Gel

Image:Asm100416.jpg

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