From 2013.igem.org
23-10 - 29-10-13
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
|
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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