~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
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
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
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
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
|
|
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
|