User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/06/02

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HCO/LCO Adenita Culture Re-Amp PCR Purification with QIAquick

For failed sequencing samples 8, 12, G

http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickpcrpurificationkit.aspx#Tabs=t2

    • see page 19.
    • 16ul PCR product left for cleaning. Resuspended in 50ul EB buffer.
Sample ID Nucleic Acid Conc. Unit A260 A280 260/280 260/230 Sample Type Factor
8 5.9 ng/µl 0.117 0.036 3.22 2.62 DNA 50
12 5.7 ng/µl 0.114 0.064 1.77 1.93 DNA 50
G 3.7 ng/µl 0.073 0.029 2.55 19.45 DNA 50

Use 4.0ul = 15-20ng for Cycle Sequencing

HCO/LCO Adenita Culture Cycle Sequencing

For failed sequencing samples 8, 12, G

AMP Lane Template ' Sample Number Primer
1 E. Windsor B2 8 HCO
2 Ithaca 1 A1 12 HCO
3 Woods Hole 1 C4 G HCO
4 E. Windsor B2 8 LCO
5 Ithaca 1 A1 12 LCO
6 Woods Hole 1 C4 G LCO
CS Reaction mixture conc. 1x 3 '
Big Dye Terminator 0.5 1.5
Primer (FWD or RVS) 5uM 0.4 1.2
DMSO 0.1 0.3
5X Seq Buffer 5X 0.4 1.2
Water 0.6 1.8
Template * ~5ng/ul 4
6 2.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C 10 sec
50C 5 sec
60C 4 min
x60