Team:Heidelberg/Templates/DelH week25

From 2013.igem.org

(Difference between revisions)
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====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)====
====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)====
 +
4 reactions with 20µl each, followed by DpnI digest
:'''Reaction'''  
:'''Reaction'''  
{| class="wikitable"
{| class="wikitable"
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| 1  || 12 || inf
| 1  || 12 || inf
|}
|}
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====Amplification of DelH fragment (FS_69 to FS_70; 7.0 kbp)====
 +
7 reactions with 20µl each
 +
:'''Reaction'''
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{| class="wikitable"
 +
|-
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! Reagent !! Amount [µl] 
 +
|-
 +
| ''D. acidovorans'' SPH-1 || 1
 +
|-
 +
| FS_69:  (1/10) || 1
 +
|-
 +
| FS_70:  (1/10) || 1
 +
|-
 +
| Phusion flash Master Mix || 10
 +
|-
 +
| DMSO || 1
 +
|-
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| dd H<sub>2</sub>O || 4
 +
|}
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 +
:'''Conditions'''
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 +
{| class="wikitable"
 +
|-
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! colspan="3" | Biorad T100
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|-
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! Cycles !! temperature [°C] !! Time [s]
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|-
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| 1 || 98 || 10
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|-
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| rowspan="3"| 12 || 98 || 1
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|-
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| 68 ↓ 0.5  || 5
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|-
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| 72  || 3:20
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|-
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| rowspan="3"| 18 || 98 || 1
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|-
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| 66  || 5
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|-
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| 72  || 3:20
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|-
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| 1 || 72 || 10min
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|-
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| 1  || 12 || inf
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|}
 +

Revision as of 15:58, 22 October 2013

Contents

23-09 - 29-09-13

Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf


Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
58 5
72 1:25
1 72 10min
1 12 inf

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
58 5
72 1:25
1 72 10min
1 12 inf

Amplification of pFS_03 Backbone (HM_17 to FS_78; 4.1 kbp)

Reaction
Reagent Amount [µl]
pSB6A1 1
HM_17: (1/10) 1
FS_78: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
58 5
72 1:25
1 72 10min
1 12 inf

Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pDonor-Plasmid 1
FS_90: (1/10) 1
FS_93: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
35 98 1
72 1:25
1 72 10min
1 12 inf

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pSB6A1 1
FS_85: (1/10) 1
FS_94: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pSB4K5 1
FS_86: (1/10) 1
FS_87: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf

Amplification of DelH fragment (FS_69 to FS_70; 7.0 kbp)

7 reactions with 20µl each

Reaction
Reagent Amount [µl]
D. acidovorans SPH-1 1
FS_69: (1/10) 1
FS_70: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 10min
1 12 inf



18.10

HM17-FS_78

Annealing temps: 62°C, 60°C, 57°C, 55°C
Elongation time: 2:10
--> Did not work

Screening remaining Gibson electroporated

60 colonies were picked and screened with primers FS_73 and FS_95 (annealing 66 touchdown, 62)

Gibson assembly

Reagent Volume added (µl)
BB (FS..) 0.61
DN11-FS66 1.00
FS_67-FS_68 1.35
FS_69-FS_70 6.34
FS_71-HM08 0.70
Gibson Master Mix 2x 10

ccdB

Annealing temps: </br> 12 cycles 74, 68, 64 and 58°C
18 cycles 72, 66, 62 and 56°C

20-10-2013

Inocultation triple dummy clone

Screening triple clone

each with positive control IK25+VF2 -->54°C, 90s elongation
FS47+FS48 --> 66t,62 40s elongation
FS95+FS73 --> 66t,62 40s elongation

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