From 2013.igem.org
07-08-2013
Amplification from FS_08 to FS_23; 6.5 kb
Amplifcation of DelAF(I), DelFG, DelG (07.08); run at 100 V, 0.8 % gel (TAE)
Amplifcation of DelAF(I), DelFG, DelG (07.08), cut
- Reaction
| what | µl
|
| D. acidovorans SPH-1 | 1
|
| FS_08: (1/10) | 2
|
| FS_23: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| dd H2O | 4
|
| DMSO | 1
|
- Conditions
| Biorad MyCycler
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 66 ↓ 0.5 | 5
|
| 72 | 3:20
|
| 18 | 98 | 1
|
| 64 | 5
|
| 72 | 3:20
|
| 1 | 72 | 10min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification from SR_01 to FS_23; 6.2 kb
Amplifcation of DelAF and DelG; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII; run at 100 V, 0.8 % gel (TAE)(07.08)
Amplifcation of DelG (SR01-FS23), pSB4K5 and DelAFII cut; run at 100 V, 0.8 % gel (TAE)(07.08)
- Reaction
| what | µl
|
| D. acidovorans SPH-1 | 1
|
| SR_01: (1/10) | 2
|
| FS_23: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 66 ↓ 0.5 | 5
|
| 72 | 3:20
|
| 18 | 98 | 1
|
| 64 | 5
|
| 72 | 3:20
|
| 1 | 72 | 10min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
08-08-2013
Restriction digest of fragment SR_01 to FS_23; 6.2 kb; 07-08-2013 with BglII
Restriction digest of DelG (SR01-FS23), DelG (FS21-FS26) and DelOP; run at 100 V, 0.8 % gel (TAE)(07.08)
Incubation at room temperature for about 4h and at 37°C for 1 hours
| what | µl
|
| SR_01 to FS_23 (07-08-2013) | 20
|
| BglII | 1
|
| Buffer 3.1 | 3
|
| dd H2O | 6
|
| Expected fragment lengths [bp] | 3415, 3115
|
Results:
- Restriction digest of DelG (SR_01 to FS_23) worked out as predicted, PCR-fragment will be sequenced before potential Gibson Assembly
Concentration measurement
| Fragment | Primer | Date PCR | Concentration
|
| DelG | SR01-FS23 | 07-08-2013 | 34.9 ng/µl
|
| DelG | FS08-FS23 | 07-08-2013 | 25.6 ng/µl
|
09-08-2013
Amplification from SR_01 to FS_23; 6.2 kb
Amplifcation of DelAF and DelG; run at 100 V, 0.8 % gel (TAE)(09.08)
- Reaction
| what | µl
|
| D. acidovorans SPH-1 | 1
|
| SR_01: (1/10) | 2
|
| FS_23: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 66 ↓ 0.5 | 5
|
| 72 | 3:20
|
| 18 | 98 | 1
|
| 64 | 5
|
| 72 | 3:20
|
| 1 | 72 | 10min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelG did not work
- Amplification will be repeated as the protocol was established and carried out successfully several times before, so a pipetting error might be the reason of the failure
"
_FS21-26(FG)_FS8-23(G)besch.png)
_FS21-26(FG)_FS8-23(G)cut.png)
_4xpSB4K5_2xFS02-FS05(AF)besch.png)
_4xpSB4K5_2xFS02-FS05(AF)_cut.png)
_FS21-FS26(ClaI)_OP(EcoRI).png)
