Alien Gene 454 Adapter Extension
EX Takara RXN Mixture
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conc
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1X
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24
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'
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'
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'
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'
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'
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'
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Milli Q water |
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6.15 |
147.6 |
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Takara EX Buffer |
10X |
1 |
24 |
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dNTP |
2.5uM |
0.8 |
19.2 |
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Primer Mix |
20uM |
1 |
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EX Taq |
5U |
0.05 |
1.2 |
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Template |
Oleg PCR 1:10 |
1 |
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PCR 1:5 |
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192 |
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8.0ul / RXN add 1.0ul Primer and 1.0ul PCR template |
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PCR cycle |
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98C |
10 sec |
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60C |
1 min |
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x15 |
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Primer |
Colonies -----> |
28 |
32 |
51 |
54 |
55 |
64 |
65 |
67 |
70
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B |
Hist F1/H |
ok = 1 |
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? = 5 |
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ok = 10 |
ok = 13 |
ok = 16 |
ok = 20
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C |
Histone 2 |
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ok = 4 |
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ok = 8 |
ok = 9 |
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ok = 17 |
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D |
HSP82 F2/R2 |
ok = 2 |
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ok = 6 |
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ok = 11 |
ok = 14 |
ok = 18 |
ok = 21
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E |
Myosin F2/R2 |
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F |
Rho |
ok = 3 |
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? = 7 |
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ok = 12 |
? = 15 |
ok = 19 |
ok = 22
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Primer Set |
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4 |
8 |
3 |
6 |
7 |
4 |
5 |
7 |
10
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Load 2.0ul on gel
Library preparation by Nextera DNA sample Prep kit
Tagmentation Reaction
Reaction mixture
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conc.
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'
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AGM |
15.6 ng/ul |
1.5
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Nextera Reaction Buffer LMW |
5x |
2
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Nextera Enzyme mix (Roche Titanium-compatible) |
?? |
0.5
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H2O |
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6
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total |
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10
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55C 5min |
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Purified by MinElute kit (qiagen) |
11 ul of buffer EB
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Addition of emPCR-Compatible Sites and Library Enrichment
Reaction mixture
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conc.
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'
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Recovered DNA fragment library |
?? |
2.5
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Nextera PCR buffer |
2x |
12.5
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Nextera Primer Cocktail |
50x |
0.5
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Nextera Adaptor1 |
50x |
0.5
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Nextera PCR enzyme |
2.5 U/ul |
0.5
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H2O |
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8.5
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total |
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25
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72C |
3 min
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95C |
30 sec
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95C |
10 sec
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55C |
30sec
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72C |
3min
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15 cycle
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laod 5.0 ul of reaction
Tagmentation Reaction
Reaction mixture
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conc.
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'
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AGM |
15.6 ng/ul |
0.75
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Nextera Reaction Buffer LMW |
5x |
2
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Nextera Enzyme mix (Roche Titanium-compatible) |
?? |
0.5
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H2O |
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6.75
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total |
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10
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55C 5min |
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Purified by MinElute kit (qiagen) |
11 ul of buffer EB
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Addition of emPCR-Compatible Sites and Library Enrichment
Reaction mixture
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conc.
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'
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Recovered DNA fragment library |
?? |
2.5
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Nextera PCR buffer |
2x |
12.5
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Nextera Primer Cocktail |
50x |
0.5
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Nextera Adaptor1 |
50x |
0.5
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Nextera PCR enzyme |
2.5 U/ul |
0.5
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H2O |
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8.5
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total |
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25
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72C |
3 min
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95C |
30 sec
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95C |
10 sec
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55C |
30sec
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72C |
3min
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18 cycle
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laod 5.0 ul of reaction
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