User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/15

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Nuclear Gene

Test amplification testing R Taq vs Top Taq

TOP TAQ RXN mixture conc. 1x 4 '
Template AV 20ng/ul 1 4
TopTaq buffer 10x 1 4
dNTP mix 10 mM each 0.2 0.8
Primer mix 5 uM each 1
TopTaq (QIAGEN) 5U/ul 0.05 0.2
Milli Q water 6.75 27
10 ul 36 9.0ul / RXN
R Takara RXN Mixture conc 1X 4
Milli Q water 5.55 22.2
Takara R Buffer 10X 1 4
MgCl2 25uM 0.6 2.4
dNTP 2.5uM 0.8 3.2
Primer Mix 5uM 1
R Taq 5U 0.05 0.2
Template 1ng / 20ng 1 4
36 9.0ul / RXN
A PCR cycle
95C 3 min
95C 5 sec
60C 1 min
x35
AMP Lane PCR Mix Template Primer
1 R Taq AV 1ng AV10027
2 R Taq AV 1ng A1
3 R Taq AV 1ng A2
4 R Taq AV 20ng AV10027
5 R Taq AV 20ng A1
6 R Taq AV 20ng A2
7 Top Taq AV 1ng AV10027
8 Top Taq AV 1ng A1
* AV10027 Positive Control


File:15April10 B.tif

CCA3 Qiagen Extraction

~100-200 individuals

Followed standard Qiagen tissue extraction protocol

Nanodrop 0.5ng/ul DNA