Team:Heidelberg/Templates/DelH week23

From 2013.igem.org

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| Primer fw 10 µM|| 2 µl FS78
| Primer fw 10 µM|| 2 µl FS78
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| Primer rev 10 µM|| 2 µl HM17
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|-
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| Phusion Flash Ready Mix || 10 µl
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|-
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| ddH<sub>2</sub>O  || 5 µl
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|}
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{| class="wikitable" style="float:left; margin-right:1em"
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|-
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! Cycles !! Temperature [°C] !! Time [s]
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| 1 || 98 || 10
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| rowspan="3" | 12 || 98 || 1
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| 68 (touchdown -0,5°C) || 5
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| 72 || 1:25
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| rowspan="3" | 18 || 98 || 1
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| 68 || 5
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| 72 || 1:25
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| 1 || 72 || 5 min
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| 1  || 4 || inf
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|}
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{| class="wikitable" style="float:left; margin-right:1em"
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|-
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! Cycles !! Temperature [°C] !! Time [s]
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| 1 || 98 || 5
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| rowspan="3" | 12 || 98 || 1
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| 58 || 5
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| 72 || 1:25
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| 1 || 72 || 5 min
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| 1  || 4 || inf
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====Amplification of backbone pSB6A1 for strategy pFS02====
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{| class="wikitable" style="float:left; margin-right:1em"
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! Reagent !! Amount
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| Template ||  1 µl of minipreped pSB6A1 (2L)
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|-
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| Primer fw 10 µM|| 2 µl FS85
|-
|-
| Primer rev 10 µM|| 2 µl HM17
| Primer rev 10 µM|| 2 µl HM17

Revision as of 13:42, 22 October 2013

Contents

30-09 - 06-10-13

Generation of DelH Plasmid pHM04 15-09

Colony-PCR Conditions CP.W23.A

Template 23x 1 picked colony, positive control
Expected length [bp] 663
Named 1A - 12H
Primer fw 10 µM 100x 2 µl VF2
Primer rev 10 µM 100x 2 µl DN07
iTaq Polymerase (2x) 100x 10 µl
ddH2O 96x 6 µl
Cycles Temperature DelH [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45
18 95 30
65 30
72 45
1 12 inf

Result

Expected band: 663 bp
[[|200px|thumb|right|Fig.23.1 Colony PCR of Gibson assembled DelH-BB without mRFP 23 colonies]]


Amplification of backbone pSB6A1 for strategy pFS02

Reagent Amount
Template 1 µl of minipreped pSB6A1 (2L)
Primer fw 10 µM 2 µl FS78
Primer rev 10 µM 2 µl HM17
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
68 (touchdown -0,5°C) 5
72 1:25
18 98 1
68 5
72 1:25
1 72 5 min
1 4 inf
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
58 5
72 1:25
1 72 5 min
1 4 inf

Amplification of backbone pSB6A1 for strategy pFS02

Reagent Amount
Template 1 µl of minipreped pSB6A1 (2L)
Primer fw 10 µM 2 µl FS85
Primer rev 10 µM 2 µl HM17
Phusion Flash Ready Mix 10 µl
ddH2O 5 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
68 (touchdown -0,5°C) 5
72 1:25
18 98 1
68 5
72 1:25
1 72 5 min
1 4 inf
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
58 5
72 1:25
1 72 5 min
1 4 inf
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