User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/13

Alien genes

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Amplifying gene rich region of CCA3 Using AV as positive control for primers PCR conditions Reaction mixture conc. 1x 17 water 5.35 90.95 LA buffer 10x 1 17 dNTP mix 2.5 mM each 1.6 27.2 Primer mix 5 uM each 1 TakaraLA 5U/ul 0.05 0.85 template 1 ng/ul 1 8 Primer sets (first row of diluted plate): Avhsp-1_1-1F Avhsp-1_4-1F Avhsp-1_7-1F Avhsp-3_3-1F Avhsp-4_3-1F Avhis-1_2-1F Avhis-2_2-1F Avhis-3_2-1F 94C,1 min (98C,10 sec;65C 10 min)x30 Gel Gene-rich region of CC A3 - the rest of the primers Program Reaction mixture conc. 1x 44 water 5.35 235.4 LA buffer 10x 1 44 dNTP mix 2.5 mM each 1.6 70.4 Primer mix 5 uM each 1 TakaraLA 5U/ul 0.05 2.2 template 1 ng/ul 1 44 8 primer orientation ' 1 2 3 4 5 6 7 8 a Avhsp-1_1-2F Avhsp-1_4-2F Avhsp-1_7-2F Avhsp-3_3-2F Avhsp-4_3-2F Avhis-1_2-2F Avhis-2_2-2F Avhis-3_2-2F b Avhsp-1_2-1R Avhsp-1_5-1R Avhsp-3_1-1R Avhsp-4_1-1R Avhsp-4_4-1R Avhis-1_3-1R Avhis-2_3-1R Avhis-3_3-1R c Avhsp-1_2-2F Avhsp-1_5-2F Avhsp-3_1-2F Avhsp-4_1-2F Avhsp-4_4-2F Avhis-1_3-2F Avhis-2_3-2F Avhis-3_3-2F d Avhsp-1_3-1F Avhsp-1_6-1F Avhsp-3_2-1F Avhsp-4_2-1F Avhis-1_1-1F Avhis-2_1-1F Avhis-3_1-1F Avhsp-4_1-1F e Avhsp-1_3-2F Avhsp-1_6-2F Avhsp-3_2-2F Avhsp-4_2-2F Avhis-1_1-2F Avhis-2_1-2F Avhis-3_1-2F Avhsp-4_1-2F f Avhsp-4_2-1F Avhsp-4_2-2F 94C,1 min (98C,10 sec;65C 10 min)x30 Gel Alien Genes New Primer Screen Reaction mixture conc. 1x x16.5 ' lane ' Temp ?? 2 27 Ithaca 2 A3 TopTaq Buffer 10x 1 16.5 28 Ithaca 2 A4 dNTP mix 10 mM each 0.2 3.3 29 Ithaca 2 A5 Primer mix (HCO/LCO) 5 uM each 1 16.5 37 Tsukuba 3 D2 TopTaq 5 U/ul 0.05 0.825 40 Woods Hole 1 A6 Milli Q 5.75 94.875 43 Yokohama B5 10 ul 47 Harvard A1 48 Ithaca 2 A6 PCR cycle 50 Tsukuba 1 B5 94C 3 min 51 Yokohama A2 55 Yokohama B2 98C 10 sec 56 Yokohama A4 60C 3 min 61 Pennsylvania(PA) B3 x35 62 Ithaca1 C3 75 Baltimore A5 72C 5 min 76 Pennsylvania (PA) B1