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Team:Heidelberg/Templates/DelH week25
From 2013.igem.org
(Difference between revisions)
| Line 532: | Line 532: | ||
|- | |- | ||
| ddH<sub>2</sub>O || 60x 4 µl | | ddH<sub>2</sub>O || 60x 4 µl | ||
| + | |} | ||
| + | {| class="wikitable" style="float:left; margin-right:1em" | ||
| + | |- | ||
| + | ! Cycles !! Temperature [°C] !! Time [s] | ||
| + | |- | ||
| + | | 1 || 95 || 120 | ||
| + | |- | ||
| + | | rowspan="3" | 12 || 95 || 30 | ||
| + | |- | ||
| + | | 68 (touchdown -0.5°C) || 30 | ||
| + | |- | ||
| + | | 72 || 45 s | ||
| + | |- | ||
| + | | rowspan="3" | 18 || 95 || 30 | ||
| + | |- | ||
| + | | 65 || 30 | ||
| + | |- | ||
| + | | 72 || 45 | ||
| + | |- | ||
| + | | 1 || 12 || inf | ||
| + | |} | ||
| + | <div style="clear:both"></div> | ||
| + | |||
| + | ==Gibson assembly of pFS_04== | ||
| + | {|class="wikitable" | ||
| + | |- | ||
| + | ! Reagent !! Volume added (µl) | ||
| + | |- | ||
| + | | BB (FS_85-FS_94) || 0.44 | ||
| + | |- | ||
| + | | ccdB (FS_90-FS_93 || 0.15 | ||
| + | |- | ||
| + | | ddH<sub>2</sub>O || 9.41 | ||
| + | |- | ||
| + | | Gibson Master Mix 2x || 10 | ||
| + | |} | ||
| + | |||
| + | |||
| + | ====Colony-PCR, screening for pFS_04==== | ||
| + | {| class="wikitable" style="float:left; margin-right:1em" | ||
| + | |- | ||
| + | | 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 | ||
| + | |- | ||
| + | | ddH<sub>2</sub>O || 30x 4 µl | ||
|} | |} | ||
{| class="wikitable" style="float:left; margin-right:1em" | {| class="wikitable" style="float:left; margin-right:1em" | ||
Revision as of 14:08, 25 October 2013
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 |
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 |
| 12 | 95 | 30 |
| 68 (touchdown -0.5°C) | 30 | |
| 72 | 45 s | |
| 18 | 95 | 30 |
| 65 | 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
