User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/16

Alien genes

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CCA3 Qiagen Extraction ~100-200 individuals Used 1mM NaCl to make rotifers curl, then moved to extraction tube Followed standard Qiagen tissue extraction protocol Nanodrop DNA Tube (1) 6.9 ng/ul Tube (2) 3.1 ng/ul R Takara Test Nuclear Genes R Takara RXN Mixture conc 1X 6.5 ' Milli Q water 4.55 29.575 Takara R Buffer 10X 1 6.5 MgCl2 25uM 0.6 3.9 dNTP 2.5uM 0.8 5.2 Primer Mix 5uM 1 R Taq 5U 0.05 0.325 Template instagene 2 13 58.5 9.0ul / RXN A PCR cycle 95C 3 min 95C 5 sec 60C 1 min x35 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 * AV Positive Control Load 2.0ul sample R Takara Test and HS Takara Test Nuclear Genes R Takara RXN Mixture conc 1X 6.5 ' Milli Q water 4.55 29.575 Takara R Buffer 10X 1 6.5 MgCl2 25uM 1.2 7.8 dNTP 2.5uM 0.8 5.2 Primer Mix 5uM 1 R Taq 5U 0.05 0.325 Template Instagene 2 13 62.4 9.0ul / RXN 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 95C 3 min 95C 5 sec 60C 1 min 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 PCR test (TopTaq) Reaction mixture conc. 1x x3.5 ' lane ' Temp (InstaGene) ?? 2 1 A.vaga TopTaq Buffer 10x 1 3.5 2 CC A3 dNTP mix 10 mM each 0.2 0.7 3 Tsukuba D4 (negative control) Primer mix (AV10027) 5 uM each 1 3.5 TopTaq 5 U/ul 0.05 0.175 Milli Q 5.75 20.125 10 ul PCR cycle 94C 3 min 98C 10 sec 60C 3 min x35 72C 5 min load 2.5ul of sample Alien Genes New Primer Screen Reaction mixture conc. 1x 32.5 ' Lane Gene Lane Gene Temp (Tsukuba D4, InstaGene) ?? 2 65 1 AV10158 17 AV10084 TopTaq Buffer 10x 1 32.5 2 AV10153 18 AV10082 dNTP mix 10 mM each 0.2 6.5 3 AV10151 19 AV10075 Primer mix 5 uM each 1 4 AV10150 20 AV10071 TopTaq 5 U/ul 0.05 1.625 5 AV10140 21 AV10045 Milli Q 5.75 186.875 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 94C 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 sample Gene-rich region of CCA3 -- take 2 this time going to try the same thing but with higher DNA concentration PCR conditions Reaction mixture conc. 1x 12.5 water 5.35 66.875 LA buffer 10x 1 12.5 dNTP mix 2.5 mM each 1.6 20 Primer mix 5 uM each 1 TakaraLA 5U/ul 0.05 0.625 template 3 ng/ul 1 12.5 9 Primers: 1. Avhsp-1_2-1R 7. Avhsp-3_1-2F 2. Avhsp-1_3-2F 8. Avhsp-4_1-2F 3. Avhsp-1_6-1F 9. Avhsp-4_3-1F 4. Avhsp-1_7-1F 10. Avhis-2_2-2F 5. Avhsp-1_7-2F 11. Avhsp-4_2-1F 6. Avhsp-3_1-1R 12. Avhsp-4_2-2F 94C 1 min, (98C, 10 sec;65C 10 min)x30, 72C 5 min Gel