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
|
The colony PCR was positive for several clones. Those clones were cultivated and preped.
Restriction Digest with PvuI
The restriction digest was positive for colonies B9 and B10.
Sequencing of clones B9 and B10 with DN07
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