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
|