User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/10

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Nuclear Region HS Takara and EX Takara PCR Optimization and Test New 20uM Primer Dilution

HS Takara RXN Mixture conc 1X 3 '
Milli Q water 3.1 9.3
Takara HS Buffer 5X 2 6
dNTP 2.5uM 0.8 2.4
Primer Mix 20uM 1
HS Taq 5U 0.1 0.3
Template Instagene 3 9
27 9.0ul / RXN
add 1.0ul Primer
EX Takara RXN Mixture conc 1X 3
Milli Q water 4.15 12.45
Takara EX Buffer 10X 1 3
dNTP 2.5uM 0.8 2.4
Primer Mix 20uM 1
EX Taq 5U 0.05 0.15
Template Instagene 3 9
27 9.0ul / RXN
add 1.0ul Primer
A PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Primer Amt of Template Taq
1 3 Instagene B2 3 HS
2 4 Instagene B2 3 HS
3 AV 20ng B2 1 HS
4 3 Instagene B2 3 EX
5 4 Instagene B2 3 EX
6 AV 20ng B2 1 EX
*AV 20ng Positive Control

Load 2.0ul Sample

10May10.jpg

HS failed. ---> Test EX

Nuclear Region 454 Adenita Culture Amplification

Test on Adenita Culture 1 and 2

EX Takara RXN Mixture conc 1X 5.5 '
Milli Q water 4.15 22.825
Takara EX Buffer 10X 1 5.5
dNTP 2.5uM 0.8 4.4
Primer Mix 20uM 1
EX Taq 5U 0.05 0.275
Template Instagene 3 16.5
49.5 9.0ul / RXN
add 1.0ul Primer
PCR cycle
98C 10 sec
60C 1 min
x35
AMP Lane Template Primer
1 1 Instagene B1
2 1 Instagene C1
3 1 Instagene D1
4 1 Instagene E1
5 1 Instagene F1
6 2 Instagene B2
7 2 Instagene C2
8 2 Instagene D2
9 2 Instagene E2
10 2 Instagene F2
11 AV 20ng B1
12 AV 20ng C1
13 AV 20ng D1
14 AV 20ng E1
15 AV 20ng F1
*AV 20ng Positive Control

Load 2.0ul Sample

10May10B.jpg

Verification of TopTaq buffer

Reaction mixture conc. 1x x8.5x2 ' No. Primer mix ' '
Temp (Tsukuba3 D4 (InstaGene)) ?? 2 17 1 AV10134 9 AV10134
TopTaq Buffer 10x 1 8.5 2 AV10121 10 AV10121
dNTP mix 10 mM each 0.2 1.7 3 AV10084 11 AV10084
Primer mix 5 uM each 1 4 AV10045 12 AV10045
TopTaq 5 U/ul 0.05 0.425 5 AV10044 13 AV10044
Milli Q 5.75 48.875 6 AV10034 14 AV10034
10 ul 7 AV10027 15 AV10027
8 AV10025 16 AV10025
PCR cycle Old buffer 17 New buffer
94C 3 min
98C 10 sec
60C 3 min
x35
72C 5 min

load 2.5 ul of sample

0096.jpg