From 2013.igem.org
13-08-2013
Amplification from FS_21 to FS_07; 5.2 kb
Amplifcation of DelFG (13-08); run at 100 V, 0.8 % gel (TAE)
- Reaction
| Reagent | µl
|
| Template | D.acidovorans SPH-1 colony
|
| Primer fw | 4 µl FS_21
|
| Primer rev | 4 µl FS_07
|
| Phusion Ready Mix | 10 µl
|
| DMSO | 1 µl
|
| dd H2O | 1 µl
|
- Conditions
| Biometra TProfessional Basic
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 55 | 2:00
|
| 1 | 72 | 10 min
|
| 1 | 10 | inf
|
Results:
- Amplification of DelFG worked, but amount of DNA was insufficient
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be carried out with another cylcer which by experience of the last amplifications led to higher product yields.
15-08-2013
Amplification from FS_21 to FS_26 ; 5.5 kb
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
- Reaction
(5x20 µl)
| what | µl
|
| D. acidovorans SPH-1 colony | 1
|
| FS_21: (1/10) | 2
|
| FS_26: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biometra TProfessional Basic
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 60 | 5
|
| 72 | 2:15
|
| 1 | 72 | 10 min
|
| 1 | 10 | inf
|
Results:
- Amplification of DelFG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- obtained DNA will consequently be used for Gibson Assembly
- PCR will be repeated to get higher amounts of DNA and increase concentration by purifying several gel slices using the same QIAquick spin column
16-08-2013
Concentration measurement DelFG
all fragments were gel purified, FG were additionally purified with the nucleotide removal kit
| Fragment | Primer | Date PCR | Concentration
|
| DelFG | FS21-FS26 | 29-07-2013 | 61.4 ng/µl
|
| DelFG after nucleotide removal | FS21-FS26 | 29-07-2013 | 30 ng/µl
|
Amplification from FS_21 to FS_26 ; 5.5 kb
Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG ; run at 100 V, 0.8 % gel (TAE)(14.08)
- Reaction
(5x20 µl)
| what | µl
|
| D. acidovorans SPH-1 colony | 1
|
| FS_21: (1/10) | 2
|
| FS_26: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 4
|
- Conditions
| Biometra TProfessional Basic
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 60 | 5
|
| 72 | 2:15
|
| 1 | 72 | 10 min
|
| 1 | 10 | inf
|
Results:
- Amplification of DelFG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- obtained DNA will consequently be used for Gibson Assembly
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