From 2013.igem.org
25-07-2013
Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013
Re-PCR I of OP (FS_22 to FS_13_long); run at 100 V, 0.8 % gel (TAE)
3x20µl
- Reaction
| what | µl
|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1
|
| FS_22: (1/10) | 4
|
| FS_13_long: (1/10) | 4
|
| Phusion flash Master Mix | 10
|
| dd H2O | 1
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 65 ↓ 0.5 | 5
|
| 72 | 1:00
|
| 18 | 98 | 1
|
| 63 | 5
|
| 72 | 1:00
|
| 1 | 72 | 5min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelOP led to a smear and several unexpected bands
- Re-PCR will be repeated
Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013
4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07); run at 100 V, 0.8 % gel (TAE)
4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07) after cutting; run at 100 V, 0.8 % gel (TAE)
3x20µl
- Reaction
| what | µl
|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1
|
| FS_22: (1/10) | 2
|
| FS_13_long: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| dd H2O | 5
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 65 ↓ 0.5 | 5
|
| 72 | 1:00
|
| 18 | 98 | 1
|
| 63 | 5
|
| 72 | 1:00
|
| 1 | 72 | 5min
|
| 1 | 12 | inf
|
Results:
- a smear as well as a band of the expeceted size occured
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- DNA will be used for restriction digests to validate amplicon
26-07-2013
Restriction digest of fragment FS_22 to FS_13s; 2.7 kb; 21-07-2013 with EcoRI-HF
Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)
Incubation at 37°C for 1h 45min
| what | µl
|
| FS_22 to FS_13short (21-07-2013) | 20
|
| EcorRI-HF | 0.8
|
| CutSmart | 2.5
|
| dd H2O | 2.5
|
| Expected fragment lengths [bp] | 1883, 960
|
Results:
- Restriction digest did not work, the amplified fragment was either not cut or the fragments are not visible due to a very small amount of DNA
- Re-PCR will be repeated to obtain the higher amounts of DNA required for the restriction digest
30-07-2013
Re-PCR from FS_22 to FS_13; 2.7 kb; 19-07-2013
Re-PCR OP, Restriction digest OP with EcoRI; run at 100 V, 0.8 % gel (TAE)
3x20µl
- Reaction
| what | µl
|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1
|
| FS_22: (1/10) | 2
|
| FS_13_long: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| dd H2O | 5
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 65 ↓ 0.5 | 5
|
| 72 | 1:00
|
| 18 | 98 | 1
|
| 63 | 5
|
| 72 | 1:00
|
| 1 | 72 | 5min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelOP did not work
- PCR will be repeated with different primer concentrations to estimate whether primer dimes might be the reason for insufficient amplification
Restriction digest from FS_22 to FS_13; 2.7 kb; 25-07-2013 with EcoRI-HF
Incubation at 37°C for
| what | µl
|
| FS_22 to FS_13long(25-07-2013) | 17
|
| EcoRI | 1
|
| Buffer CutSmart | 2
|
Amplification from FS_22 to FS_13s; 2.7 kb
Amplification of Del O-P from FS_22 to FS_13long/FS_13short under different conditions run at 100 V 0.8% agarose gel (TAE
2x20µl
- Reaction
| what | µl
|
| D.acidovorans DSM-39 | 1
|
| FS_22: (1/10) | 4
|
| FS_13_short: (1/10) | 4
|
| Phusion flash Master Mix | 10
|
| dd H2O | 1
|
20µl
- Reaction
| what | µl
|
| D.acidovorans DSM-39 | 1
|
| FS_22: (1/10) | 2
|
| FS_13_short: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| dd H2O | 5
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 65 ↓ 0.5 | 5
|
| 72 | 1:00
|
| 18 | 98 | 1
|
| 63 | 5
|
| 72 | 1:00
|
| 1 | 72 | 5min
|
| 1 | 12 | inf
|
Results:
- Amplification of DelOP resulted in a small band atthe desired lenght, but also a smear and several unexpected bands
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- amplicon will be used for restriction digest to validate the construct
Amplification from FS_22 to FS_13(s/l); 2.7 kb
- Reaction
| what | µl
|
| D.acidovorans DSM-39 | 1
|
| FS_22: (1/10) | 2
|
| FS_13_short/FS_13_long: (1/10) | 2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1/-
|
| dd H2O | 4/5
|
- Conditions
| Biorad C1000 Touch
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 72 | 1:00
|
| 1 | 72 | 5 min
|
| 1 | 12 | inf
|
- Conditions
| Biorad MyCycler*
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 70 | 5
|
| 72 | 1:00
|
| 1 | 72 | 5 min
|
| 1 | 12 | inf
|
"
_3xFS22toFS13.png)
_3xFS22toFS13_cut.png)
