Team:Heidelberg/Templates/DelH week24
From 2013.igem.org
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== 23-09 - 29-09-13 == | == 23-09 - 29-09-13 == | ||
===Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05=== | ===Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05=== | ||
Line 5: | Line 6: | ||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
- | ! Reagent !! Amount | + | ! Reagent !! Amount [µl] |
|- | |- | ||
| pSB6A1 || 1 | | pSB6A1 || 1 | ||
Line 12: | Line 13: | ||
|- | |- | ||
| FS_94: (1/10) || 1 | | FS_94: (1/10) || 1 | ||
+ | |- | ||
+ | | Phusion flash Master Mix || 10 | ||
+ | |- | ||
+ | | DMSO || 1 | ||
+ | |- | ||
+ | | dd H<sub>2</sub>O || 4 | ||
+ | |} | ||
+ | |||
+ | :'''Conditions ''' | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! colspan="3" | Biorad T100 | ||
+ | |- | ||
+ | ! Cycles !! temperature [°C] !! Time [s] | ||
+ | |- | ||
+ | | 1 || 98 || 10 | ||
+ | |- | ||
+ | | rowspan="2"| 35 || 98 || 1 | ||
+ | |- | ||
+ | | 72 || 1:25 | ||
+ | |- | ||
+ | | 1 || 72 || 10min | ||
+ | |- | ||
+ | | 1 || 12 || inf | ||
+ | |} | ||
+ | |||
+ | '''Results:''' | ||
+ | |||
+ | ====Amplification of pFS_02 Backbone (HM_17 to FS_78; 4.1 kbp)==== | ||
+ | :'''Reaction''' | ||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! Reagent !! Amount [µl] | ||
+ | |- | ||
+ | | pSB6A1 || 1 | ||
+ | |- | ||
+ | | HM_17: (1/10) || 1 | ||
+ | |- | ||
+ | | FS_78: (1/10) || 1 | ||
+ | |- | ||
+ | | Phusion flash Master Mix || 10 | ||
+ | |- | ||
+ | | DMSO || 1 | ||
+ | |- | ||
+ | | dd H<sub>2</sub>O || 4 | ||
+ | |} | ||
+ | |||
+ | :'''Conditions ''' | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! colspan="3" | Biorad T100 | ||
+ | |- | ||
+ | ! Cycles !! temperature [°C] !! Time [s] | ||
+ | |- | ||
+ | | 1 || 98 || 10 | ||
+ | |- | ||
+ | | rowspan="2"| 35 || 98 || 1 | ||
+ | |- | ||
+ | | 72 || 1:25 | ||
+ | |- | ||
+ | | 1 || 72 || 10min | ||
+ | |- | ||
+ | | 1 || 12 || inf | ||
+ | |} | ||
+ | |||
+ | '''Results:''' | ||
+ | |||
+ | ====Amplification of pFS_02 Backbone (HM_17 to FS_85; 4.1 kbp)==== | ||
+ | :'''Reaction''' | ||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! Reagent !! Amount [µl] | ||
+ | |- | ||
+ | | pSB6A1 || 1 | ||
+ | |- | ||
+ | | HM_17: (1/10) || 1 | ||
+ | |- | ||
+ | | FS_85: (1/10) || 1 | ||
+ | |- | ||
+ | | Phusion flash Master Mix || 10 | ||
+ | |- | ||
+ | | DMSO || 1 | ||
+ | |- | ||
+ | | dd H<sub>2</sub>O || 4 | ||
+ | |} | ||
+ | |||
+ | :'''Conditions ''' | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! colspan="3" | Biorad T100 | ||
+ | |- | ||
+ | ! Cycles !! temperature [°C] !! Time [s] | ||
+ | |- | ||
+ | | 1 || 98 || 10 | ||
+ | |- | ||
+ | | rowspan="2"| 35 || 98 || 1 | ||
+ | |- | ||
+ | | 72 || 1:25 | ||
+ | |- | ||
+ | | 1 || 72 || 10min | ||
+ | |- | ||
+ | | 1 || 12 || inf | ||
+ | |} | ||
+ | |||
+ | '''Results:''' | ||
+ | |||
+ | ====Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)==== | ||
+ | :'''Reaction''' | ||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! Reagent !! Amount [µl] | ||
+ | |- | ||
+ | | pSB4K5 || 1 | ||
+ | |- | ||
+ | | FS_86: (1/10) || 1 | ||
+ | |- | ||
+ | | FS_87: (1/10) || 1 | ||
+ | |- | ||
+ | | Phusion flash Master Mix || 10 | ||
+ | |- | ||
+ | | DMSO || 1 | ||
+ | |- | ||
+ | | dd H<sub>2</sub>O || 4 | ||
+ | |} | ||
+ | |||
+ | :'''Conditions ''' | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! colspan="3" | Biorad T100 | ||
+ | |- | ||
+ | ! Cycles !! temperature [°C] !! Time [s] | ||
+ | |- | ||
+ | | 1 || 98 || 10 | ||
+ | |- | ||
+ | | rowspan="2"| 35 || 98 || 1 | ||
+ | |- | ||
+ | | 72 || 1:25 | ||
+ | |- | ||
+ | | 1 || 72 || 10min | ||
+ | |- | ||
+ | | 1 || 12 || inf | ||
+ | |} | ||
+ | |||
+ | '''Results:''' | ||
+ | |||
+ | ====Amplification of DelH fragment (FS_84 to FS_66; 4.1 kbp)==== | ||
+ | :'''Reaction''' | ||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! Reagent !! Amount [µl] | ||
+ | |- | ||
+ | | ''D. acidovorans'' SPH-1 || 1 | ||
+ | |- | ||
+ | | FS_86: (1/10) || 1 | ||
+ | |- | ||
+ | | FS_87: (1/10) || 1 | ||
+ | |- | ||
+ | | Phusion flash Master Mix || 10 | ||
+ | |- | ||
+ | | DMSO || 1 | ||
+ | |- | ||
+ | | dd H<sub>2</sub>O || 4 | ||
+ | |} | ||
+ | |||
+ | :'''Conditions ''' | ||
+ | |||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! colspan="3" | Biorad T100 | ||
+ | |- | ||
+ | ! Cycles !! temperature [°C] !! Time [s] | ||
+ | |- | ||
+ | | 1 || 98 || 10 | ||
+ | |- | ||
+ | | rowspan="2"| 35 || 98 || 1 | ||
+ | |- | ||
+ | | 72 || 1:25 | ||
+ | |- | ||
+ | | 1 || 72 || 10min | ||
+ | |- | ||
+ | | 1 || 12 || inf | ||
+ | |} | ||
+ | |||
+ | '''Results:''' | ||
+ | |||
+ | ====Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)==== | ||
+ | :'''Reaction''' | ||
+ | {| class="wikitable" | ||
+ | |- | ||
+ | ! Reagent !! Amount [µl] | ||
+ | |- | ||
+ | | pDonor-Plasmid || 1 | ||
+ | |- | ||
+ | | FS_90: (1/10) || 1 | ||
+ | |- | ||
+ | | FS_93: (1/10) || 1 | ||
|- | |- | ||
| Phusion flash Master Mix || 10 | | Phusion flash Master Mix || 10 |
Latest revision as of 15:35, 22 October 2013
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 |
35 | 98 | 1 |
72 | 1:25 | |
1 | 72 | 10min |
1 | 12 | inf |
Results:
Amplification of pFS_02 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 |
72 | 1:25 | |
1 | 72 | 10min |
1 | 12 | inf |
Results:
Amplification of pFS_02 Backbone (HM_17 to FS_85; 4.1 kbp)
- Reaction
Reagent | Amount [µl] |
---|---|
pSB6A1 | 1 |
HM_17: (1/10) | 1 |
FS_85: (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 |
Results:
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 |
72 | 1:25 | |
1 | 72 | 10min |
1 | 12 | inf |
Results:
Amplification of DelH fragment (FS_84 to FS_66; 4.1 kbp)
- Reaction
Reagent | Amount [µl] |
---|---|
D. acidovorans SPH-1 | 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 |
72 | 1:25 | |
1 | 72 | 10min |
1 | 12 | inf |
Results:
Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)
- 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 |
Results: