User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/14

Alien genes

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Nuclear Gene Amplification of Adenita Cultures Reaction mixture conc. 1x 6.5 ' Template Instagene ? 1 6.5 TopTaq buffer 10x 1 6.5 dNTP mix 10 mM each 0.2 1.3 Primer mix 5 uM each 1 TopTaq (QIAGEN) 5U/ul 0.05 0.325 Milli Q water 6.75 43.875 10 ul 58.5 9.0ul / RXN PCR cycle 95C 3 min 95C 5 sec 60C 1 min x35 AMP Lane Template Primer 1 71 A1 2 71 A2 3 71 A3 4 71 A4 5 71 A5 6 71 A6 7 72 B1 8 72 B2 9 72 B3 10 72 B4 11 72 B5 12 72 B6 Nuclear Gene Troubleshoot PCR Program Reaction 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 A PCR cycle 95C 3 min 95C 5 sec 60C 1 min x35 B PCR cycle 95C 3 min 95C 5 sec 60C 1 min x10 95C 5 sec 68C 1 min x25 AMP Lane Template Primer PCR Program 1 AV A1 A 2 AV *AV10027 A 3 AV A1 B 4 AV *AV10027 B * AV10027 Positive Control Load 4.0ul of sample Genome extraction Tsukuba3 D4(QIAamp DNA micro kit) 100ul EB (3.4 ug/ul) load 1.0ul of extraction Alien Genes New Primer Screen Reaction mixture conc. 1x x33 ' Lane Gene Lane Gene Temp (Tsukuba3 D4) 1ug/ul 1 33 1 AV10158 17 AV10084 TopTaq Buffer 10x 1 33 2 AV10153 18 AV10082 dNTP mix 10 mM each 0.2 6.6 3 AV10151 19 AV10075 Primer mix 5 uM each 1 4 AV10150 20 AV10071 TopTaq 5 U/ul 0.05 1.65 5 AV10140 21 AV10045 Milli Q 6.75 222.75 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 98C 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 sampple