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
85.9ng/µl0.1170.0363.222.62DNA50
125.7ng/µl0.1140.0641.771.93DNA50
G3.7ng/µl0.0730.0292.5519.45DNA50

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
1E. WindsorB28HCO
2Ithaca 1A112HCO
3Woods Hole 1C4GHCO
4E. WindsorB28LCO
5Ithaca 1A112LCO
6Woods Hole 1C4GLCO
CS Reaction mixture conc. 1x 3 '
Big Dye Terminator0.51.5
Primer (FWD or RVS)5uM0.41.2
DMSO0.10.3
5X Seq Buffer5X0.41.2
Water0.61.8
Template *~5ng/ul4
62.0ul / RXN
*QIAquick Cleaned PCR product
Cycle Seq Program
96C10 sec
50C5 sec
60C4 min
x60
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