From 2013.igem.org
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 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
|
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
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