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

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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

18May10.jpg

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.