User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/05/18

Alien genes

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HCO/LCO Adenita Culture Re-Amp For failed sequencing samples 9, 11, 13, 14, 15, 41, 46, A, CCA3 EX Takara RXN Mixture conc 1X 11 ' Milli Q water 10.3 113.3 Takara EX Buffer 10X 2 22 dNTP 2.5uM 1.6 17.6 Primer Mix 20uM 2 22 EX Taq 5U 0.1 1.1 Template Instagene 4 176 16.0ul / RXN add 4.0ul Template PCR cycle 94C 3 min 94C 10 sec 45-68C 3 min RAMP 0.5C/sec x35 68C 5 min AMP Lane Template ' Sample Number 1 E. Windsor C2 9 2 Harvard A3 11 3 Ithaca 1 A2 13 4 Ithaca 1 A3 14 5 Ithaca 1 A5 15 6 Woods Hole 1 C2 41 7 E. Windsor D4 46 8 Boston A1 A 9 Cape Cod (CC) A3 CCA3 10 AV Positive Control 11 Neg Control Load 2.0ul Sample HCO/LCO Adenita Culture Re-Amp PCR Purification with QIAquick For failed sequencing samples 9, 11, 13, 14, 15, 41, 46, A, CCA3 http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2 see page 19. 18ul PCR product left for cleaning. Resuspended in 50ul EB buffer. NanoDrop results # Sample ID Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor 1 9 9.5 ng/µl 0.19 0.092 2.06 3.48 DNA 50 2 11 10.5 ng/µl 0.21 0.125 1.68 2.63 DNA 50 3 13 10 ng/µl 0.201 0.134 1.49 2.44 DNA 50 4 14 9.6 ng/µl 0.192 0.116 1.65 2.55 DNA 50 5 15 10.8 ng/µl 0.216 0.136 1.59 2.77 DNA 50 6 41 9.6 ng/µl 0.192 0.116 1.66 1.91 DNA 50 7 46 9.1 ng/µl 0.181 0.108 1.68 2.77 DNA 50 8 A 10.1 ng/µl 0.202 0.104 1.93 3.28 DNA 50 9 CCA3 12.5 ng/µl 0.249 0.138 1.81 3.16 DNA 50 HCO/LCO Adenita Culture Cycle Sequencing For failed sequencing samples 9, 11, 13, 14, 15, 41, 46, A, CCA3 CS Reaction mixture conc. 1x 10 ' Big Dye Terminator 0.5 5 Primer (FWD or RVS) 5uM 0.4 4 DMSO 0.1 1 5X Seq Buffer 5X 3 30 Template * ~10ng/ul 2 40 4.0ul / RXN *QIAquick Cleaned PCR product Cycle Seq Program 96C 10 sec 50C 5 sec 60C 4 min x60 AMP Lane Template ' Sample Number Primer 1 E. Windsor C2 9 HCO 2 Harvard A3 11 HCO 3 Ithaca 1 A2 13 HCO 4 Ithaca 1 A3 14 HCO 5 Ithaca 1 A5 15 HCO 6 Woods Hole 1 C2 41 HCO 7 E. Windsor D4 46 HCO 8 Boston A1 A HCO 9 Cape Cod (CC) A3 CCA3 HCO 10 E. Windsor C2 9 LCO 11 Harvard A3 11 LCO 12 Ithaca 1 A2 13 LCO 13 Ithaca 1 A3 14 LCO 14 Ithaca 1 A5 15 LCO 15 Woods Hole 1 C2 41 LCO 16 E. Windsor D4 46 LCO 17 Boston A1 A LCO 18 Cape Cod (CC) A3 CCA3 LCO Seq Center cleaned and load onto genetic analyzer.