User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/19

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Qiagen Extraction Tsukuba 3 D4 & E.Windsor C3

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

Followed standard Qiagen tissue extraction protocol

Nanodrop DNA Tsukuba 3 D4 = 6.9 ng/ul E.Windsor C3 = 2.9 ng/ul

HS Takara PCR Optimization Nuclear Gene

Mix A ' ' ' '
HS Takara RXN Mixture conc 1X 6.5
Milli Q water 3.1 20.15
Takara HS Buffer 10X 2 13
dNTP 2.5uM 0.8 5.2
Primer Mix 5uM 2
HS Taq 5U 0.1 0.65
Template Instagene 2 13
52 8.0ul / RXN
A PCR cycle
95C 3 min
95C 5 sec
60C 1 min
x35
*AV Positive Control
Mix B
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
93C 3 min
93C 5 sec
55C 30 sec
72C 30 sec
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

19April10.jpg

Testing new CCA3 extraction for long PCR

PCR

Reaction mixture conc. 1x 5
water 5.35 26.75
LA buffer 10x 1 5
dNTP mix 2.5 mM each 1.6 8
Primer mix 5 uM each 1
TakaraLA 5U/ul 0.05 0.25
template 3 ng/ul 1 5
9
Primers
Avhsp-1_7-1F
Avhsp-1_7-2F
Avhsp-3_1-1R
Avhsp-3_1-2F
Avhsp-4_3-1F
  • standard long PCR cycling conditions

Gel

Asm100419.jpg

  • Only Avhsp-3_1-1R amplified well, although Avhsp-1_7-2F might work in a larger volume

PCR test (TopTaq)

Reaction mixture conc. 1x x8.5 ' No. Primer mix
Temp (A. vaga (InstaGene)) ?? 2 17 1 AV10134
TopTaq Buffer 10x 1 8.5 2 AV10121
dNTP mix 10 mM each 0.2 1.7 3 AV10084
Primer mix 5 uM each 1 4 AV10045
TopTaq 5 U/ul 0.05 0.425 5 AV10044
Milli Q 5.75 48.875 6 AV10034
10 ul 7 AV10027
8 AV10025
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 2.5ul of sample

0088.jpg

Reaction mixture conc. 1x x8.5 ' No. Primer mix
Temp (Tsukuba3 D4 (InstaGene)) ?? 2 17 1 AV10134
TopTaq Buffer 10x 1 8.5 2 AV10121
dNTP mix 10 mM each 0.2 1.7 3 AV10084
Primer mix 5 uM each 1 4 AV10045
TopTaq 5 U/ul 0.05 0.425 5 AV10044
Milli Q 5.75 48.875 6 AV10034
10 ul 7 AV10027
8 AV10025
PCR cycle
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min


load 2.5ul of sample

0089.jpg