Team:Heidelberg/Templates/DelH week25

From 2013.igem.org

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==Gibson assembly of pFS_02==
==Gibson assembly of pFS_02==

Revision as of 10:43, 26 October 2013

Contents

14-10 - 20-10-13

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 1:25
18 98 1
66 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
12 98 1
68 ↓ 0.5 5
72 1:25
18 98 1
66 5
72 1:25
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 0:18
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 1:25
18 98 1
66 5
72 1:25
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 1:25
18 98 1
66 5
72 1:25
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 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf


Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction
Reagent Amount [µl]
pSB6A1 0.2
HM_17: (1/10) 1
FS_87: (1/10) 1
Phusion flash Master Mix 10
DMSO 1
dd H2O 4.8
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 1:25
18 98 1
66 5
72 1:25
1 72 10min
1 12 inf


Gibson assembly of pFS_02

Reagent Volume added (µl)
BB (HM_17-FS_78) 0.24
DN11-FS66 2.13
FS_67-FS_68 2.89
FS_69-FS_70 2.38
FS_71-HM08 2.36
Gibson Master Mix 2x 10

Colony-PCR, screening for pFS_02

Template 96x 1 picked colony
Expected length [bp] 521
Named 1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM 96x 1 µl FS_73
Primer rev 10 µM 96x 1 µl FS_95
One-Taq Polymerase (2x) 96x 5 µl
ddH2O 96x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

Gibson assembly of pFS_03

Reagent Volume added (µl)
BB (FS_86 - FS_87) 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

Colony-PCR, screening for pFS_03

Template 60x1 picked colony
Expected length [bp] 521
Named 1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM 60x 0.5 µl FS_73
Primer rev 10 µM 60x 0.5 µl FS_95
One-Taq Polymerase (2x) 60x 5 µl
ddH2O 60x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45 s
18 95 30
65 30
72 45
1 12 inf

Gibson assembly of pFS_04

Reagent Volume added (µl)
BB (FS_85-FS_94) 0.44
ccdB (FS_90-FS_93 0.15
ddH2O 9.41
Gibson Master Mix 2x 10


Colony-PCR, screening for pFS_04

Template 30x1 picked colony
Expected length [bp] ~800bp
Named 1A - 6C, pSB6A1_ccdB
Primer fw 10 µM 30x 0.5 µl KH_9
Primer rev 10 µM 30x 0.5 µl VF2
One-Taq Polymerase (2x) 30x 5 µl
ddH2O 30x 4 µl
Cycles Temperature [°C] Time [s]
1 95 120
35 95 30
57 30
72 45
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


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

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/DelH_week25"