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

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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 water 4.55 29.575
Takara R Buffer 10X 1 6.5
MgCl2 25uM 0.6 3.9
dNTP 2.5uM 0.8 5.2
Primer Mix 5uM 1
R Taq 5U 0.05 0.325
Template instagene 2 13
58.5 9.0ul / RXN
A PCR cycle
95C 3 min
95C 5 sec
60C 1 min
x35
AMP Lane Template Primer
1 71 Instagene A1
2 71 Instagene A2
3 71 Instagene A3
4 71 Instagene A4
5 71 Instagene A5
6 71 Instagene A6
7 72 Instagene A1
8 72 Instagene A2
9 72 Instagene A3
10 72 Instagene A4
11 72 Instagene A5
12 72 Instagene A6
13 AV Instagene A1
14 AV Instagene A2
15 AV Instagene A3
16 AV Instagene A4
17 AV Instagene A5
18 AV Instagene A6
* AV Positive Control

Load 2.0ul sample

File:16April10.tif

R Takara Test and HS Takara Test Nuclear Genes

R Takara RXN Mixture conc 1X 6.5 '
Milli Q water 4.55 29.575
Takara R Buffer 10X 1 6.5
MgCl2 25uM 1.2 7.8
dNTP 2.5uM 0.8 5.2
Primer Mix 5uM 1
R Taq 5U 0.05 0.325
Template Instagene 2 13
62.4 9.0ul / RXN
HS Takara RXN Mixture conc 1X 6.5
Milli Q water 4.1 26.65
Takara HS Buffer 10X 2 13
dNTP 2.5uM 0.8 5.2
Primer Mix 5uM 1
HS Taq 5U 0.1 0.65
Template Instagene 2 13
58.5 9.0ul / RXN
A PCR cycle
95C 3 min
95C 5 sec
60C 1 min
x35
*AV Positive Control
AMP Lane Template Primer
1 71 Instagene A1
2 71 Instagene A2
3 71 Instagene A3
4 71 Instagene A4
5 71 Instagene A5
6 71 Instagene A6
7 72 Instagene A1
8 72 Instagene A2
9 72 Instagene A3
10 72 Instagene A4
11 72 Instagene A5
12 72 Instagene A6
13 AV Instagene A1
14 AV Instagene A2
15 AV Instagene A3
16 AV Instagene A4
17 AV Instagene A5
18 AV Instagene A6
19 71 Instagene A1
20 71 Instagene A2
21 71 Instagene A3
22 71 Instagene A4
23 71 Instagene A5
24 71 Instagene A6
25 72 Instagene A1
26 72 Instagene A2
27 72 Instagene A3
28 72 Instagene A4
29 72 Instagene A5
30 72 Instagene A6
31 AV Instagene A1
32 AV Instagene A2
33 AV Instagene A3
34 AV Instagene A4
35 AV Instagene A5
36 AV Instagene A6

Load 2.0ul sample

File:16April10 B.tif

PCR test (TopTaq)

Reaction mixture conc. 1x x3.5 ' lane '
Temp (InstaGene) ?? 2 1 A.vaga
TopTaq Buffer 10x 1 3.5 2 CC A3
dNTP mix 10 mM each 0.2 0.7 3 Tsukuba D4 (negative control)
Primer mix (AV10027) 5 uM each 1 3.5
TopTaq 5 U/ul 0.05 0.175
Milli Q 5.75 20.125
10 ul
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min


load 2.5ul of sample

0082.jpg

Alien Genes New Primer Screen

Reaction mixture conc. 1x 32.5 ' Lane Gene Lane Gene
Temp (Tsukuba D4, InstaGene) ?? 2 65 1 AV10158 17 AV10084
TopTaq Buffer 10x 1 32.5 2 AV10153 18 AV10082
dNTP mix 10 mM each 0.2 6.5 3 AV10151 19 AV10075
Primer mix 5 uM each 1 4 AV10150 20 AV10071
TopTaq 5 U/ul 0.05 1.625 5 AV10140 21 AV10045
Milli Q 5.75 186.875 6 AV10134 22 AV10043
10 ul 7 AV10130 23 AV10042
8 AV10123 24 AV10041
PCR cycle 9 AV10121 25 AV10034
94C 3 min 10 AV10104 26 AV10028
11 AV10109 27 AV10027
94C 10 sec 12 AV10099 28 AV10025
60C 3 min 13 AV10098 29 AV10016
x35 14 AV10094 30 AV10011
15 AV10092 31 AV10007
72C 5 min 16 AV10089 32 AV10002

load 2.5ul of sample

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
water 5.35 66.875
LA buffer 10x 1 12.5
dNTP mix 2.5 mM each 1.6 20
Primer mix 5 uM each 1
TakaraLA 5U/ul 0.05 0.625
template 3 ng/ul 1 12.5
9
  • Primers:
1. Avhsp-1_2-1R 7. Avhsp-3_1-2F
2. Avhsp-1_3-2F 8. Avhsp-4_1-2F
3. Avhsp-1_6-1F 9. Avhsp-4_3-1F
4. Avhsp-1_7-1F 10. Avhis-2_2-2F
5. Avhsp-1_7-2F 11. Avhsp-4_2-1F
6. Avhsp-3_1-1R 12. Avhsp-4_2-2F
  • 94C 1 min, (98C, 10 sec;65C 10 min)x30, 72C 5 min

Gel

Asm100416.jpg