Team:Heidelberg/Delftibactin/DelRest
From 2013.igem.org
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Revision as of 03:07, 30 September 2013
Del Rest. Creating a 31 kbp plasmid.
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Methods:
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06-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
what | µL |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 1 |
FS_03: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
63 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification worked with 5% DMSO
- Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices
03-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
µL 1st PCR | what | µL 2nd PCR |
---|---|---|
1 | D. acidovorans DSM-39 | 1 |
2.5 | FS_02 (1/10) | 2.5 |
2.5 | FS_05 (1/10) | 2.5 |
25 | Phusion Master Mix | 25 |
- | DMSO | 2.5 |
19 | dd H2O | 19 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- sample has to be purified before further use since contamination with propanol was present
04-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
what | µL 2nd PCR |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02 (1/10) | 2.5 |
FS_05 (1/10) | 2.5 |
Phusion Master Mix | 25 |
DMSO | 2.5 |
dd H2O | 16.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
- PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
05-08-2013
Amplification from FS_02 to FS_07; 16.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 1 |
FS_07: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
6 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 5min 40s | |
24 | 98 | 1 |
60 | 5 | |
72 | 5min 40s | |
1 | 72 | 17 min |
1 | 4 | inf |
Results:
- amplification of DelAG did not work
- repeat PCR with higher annealing temperature to increase specifity
06-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_07: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelEG worked but another unexpected product was amplified as well
- band was cut out avoiding contamination with other product and DNA purified using QIAquick Gel Extraction Kit
05-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_08: (1/10) | 1 |
FS_11: (1/10) | 1 |
Phusion Master Mix | 10 |
dd H2O | 6 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
14 | 98 | 1 |
62 ↓ 0.5 | 5 | |
72 | 3:20 min | |
16 | 98 | 1 |
60 | 5 | |
72 | 3:20 min | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- A weak band was visible, but it was on the wrong height.
- Accidently the band was cut anyway.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
07-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_08: (1/10) | 2.5 |
FS_11: (1/10) | 2.5 |
Phusion Master Mix | 25 |
dd H2O | 19 |
DMSO | 2.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
63 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- There was no band visible on the gel.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
04-07-2013
Amplification from FS_12 to FS_13; 2.7 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_12: (1/10) | 1 |
FS_13: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 420 |
1 | 4 | inf |
Results:
- Amplification of DelOP did not work
- Amplification will be repeated to exclude that pipetting errors were the reason for the failure
04-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_14: (1/10) | 1 |
FS_15: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 7 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had a bright band on the right height.
- Band was cut out and DNA purified using QIAquick Gel Extration Kit.
06-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_14: (1/10) | 1 |
FS_15: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 42 | |
18 | 98 | 1 |
63 | 5 | |
72 | 42 | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
05-07-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
what | µl |
---|---|
Template pSB4K5 | 1 |
FS_16: (1/10) | 1 |
FS_01: (1/10) | 1 |
Phusion Master Mix | 10 |
dd H2O | 6 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
14 | 98 | 1 |
62 ↓ 0.5 | 5 | |
72 | 3:20 min | |
16 | 98 | 1 |
60 | 5 | |
72 | 3:20 min | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked well and band was at expected height. Nevertheless the gel band did not run properly on the gel.
08-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
what | µL |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 2.5 |
FS_03: (1/10) | 2.5 |
Phusion Master Mix | 25 |
DMSO | 2.5 |
dd H2O | 19 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
63 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
12-07-2013
Amplification from FS_02 to FS_07; 16.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 2 |
FS_07: (1/10) | 2 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 4 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 5:30 | |
18 | 98 | 1 |
66 | 5 | |
72 | 5:30 | |
1 | 72 | 15min |
1 | 12 | inf |
Results:
- only a smear occured, no specific product was amplified
- PCR will be repeated with a lower, constant annealing temperature
09-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_07: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions I
Cycles-PCR | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Conditions II
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
5 | 98 | 1 |
68 | 5 | |
72 | 3 min | |
25 | 98 | 1 |
72 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelEG did not work
11-07-2013
Amplification from FS_04 to FS_11s; 17.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_11_short: (1/10) | 1 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 5:30 | |
18 | 98 | 1 |
66 | 5 | |
72 | 5:30 | |
1 | 72 | 15min |
1 | 12 | inf |
Results:
- Amplification of DelEG did not work
- Experiment will be repeated with NEB Phusion II Polymerase as Phusion II is not provided as mastermix and therefore GC-buffer can be used
Amplification from FS_04 to FS_11; 17.5 kb; Phusion II
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_11_short: (1/10) | 1 |
Phusion II | 0.2 |
DNTP | 0.4 |
Buffer | 4 |
DMSO | 0.6 |
dd H2O | 11.8 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 30 |
12 | 98 | 5 |
68 ↓ 0.5 | 30 | |
72 | 8:10 | |
18 | 98 | 5 |
66 | 30 | |
72 | 8:10 | |
1 | 72 | 15min |
1 | 17 | inf |
Results:
- Amplification of DelEG did not work
- it seems not to be possible to amplify the desired 17 kbp fragent with the chosen primers and the given template, primercombination will be changed in further amplification attempts
14-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 2 |
FS_07: (1/10) | 2 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 4 |
- Conditions I
Cycler incubation room right
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Conditions II
Cycler incubation room left
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
70 ↓ 0.5 | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 12 | inf |
Results:
- Amplification of DelEG worked with a touchdown PCR starting from 70°C annealing temperature
- band was cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be repeated to increase the amount of DNA and gather the concentrations necessary for Gibson Assembly
09-07-2013
Amplification from FS_08 to FS_11_short; 6.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_08 (1/10) | 1 |
FS_11_short (1/10) | 1 |
Phusion Master Mix | 10 |
dd H2O | 6 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 2:30 | |
24 | 98 | 1 |
63 | 5 | |
72 | 2:30 | |
1 | 72 | 10min |
1 | 4 | inf |
Results:
- A weak band was visible at the right height.
- Band was cut out and DNA purified using QIAquick Gel Extration Kit.
- Concentration after gel extration was too low
- Maybe increasing the temperature further will result in higher yield.
13-07-2013
Amplification from FS_10 to FS_11s; 3.3 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_10: (1/10) | 2 |
FS_11_short: (1/10) | 2 |
Phusion flash Master Mix | 10 |
dd H2O | 4 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
70 | 5 | |
72 | 1:10 min | |
1 | 72 | 5 min |
1 | 12 | inf |
Results:
- No band was visible on the gel.
- The PCR conditions of the 09-07-2013 should be further optimized.
Amplification from FS_10 to FS_11(s); 3.3 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_10: (1/10) | 2 |
FS_11 (short or long): (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 |
68 ↓ 0.5 | 5 | |
72 | 1:10 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 1:10 min | |
1 | 72 | 5 min |
1 | 12 | inf |
Results:
- Bright bands were visible in both, the PCR with the short and the long primer.
- The PCR with the short primers worked better than the one with the long primer.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
- Concentration after gel extraction with primer FS_11short=6ng/µl in 18µl
- Concentration after gel extraction with primer FS_11long=4ng/µl in 18µl
09-07-2013
Amplification I from FS_12 to FS_13; 2.7 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_12: (1/10) | 1 |
FS_13: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 7 min |
1 | 12 | inf |
Results:
- Amplification of DelOP did not work
- PCR was repeated, Annealing was carried out as touchdown starting from 68°C
Amplification II from FS_12 to FS_13; 2.7 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_12: (1/10) | 1 |
FS_13short: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 6 |
8 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 50 | |
24 | 98 | 1 |
72 | 5 | |
72 | 50 | |
1 | 72 | 11min |
1 | 4 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated with the newly ordered short version of primer FS_13, testing different DMSO settings
10-07-2013
Amplification from FS_12 to FS_13(s); 2.7 kb
- Reaction of DelO-P (2.6 kb)
4 reactions with different conditions: with/without DMSO, short/long Primer FS13
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_12: (1/10) | 1 |
FS_13(long or short): (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions of Del O-P
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 420 |
1 | 12 | inf |
Results:
- None of the Amplification neither with primer FS_13 nor with FS_13short worked out, nevertheless did addition of DMSO change the amplification result
- PCR will be carried out as 2-step in another more precise cycler
- PCR will be set up with as touchdown PCR, annealing starting at 68°C
- Furthermore a modified version of the forward primer will be order. This primer (FS_22) includes a recently in the Del Cluster predicted promotor as well as a likewise predicted ribosome binding site
08-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_14: (1/10) | 2.5 |
FS_15: (1/10) | 2.5 |
Phusion Master Mix | 25 |
DMSO | 2.5 |
dd H2O | 16.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 42 | |
18 | 98 | 1 |
63 | 5 | |
72 | 42 | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
08-07-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(2x50µl)
what | µl |
---|---|
Template pSB4K5 | 1 |
FS_01: (1/10) | 2.5 |
FS_16: (1/10) | 2.5 |
Phusion Master Mix | 25 |
DMSO | 2.5 |
dd H2O | 16.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
14 | 98 | 1 |
62 ↓ 0.5 | 5 | |
72 | 3:20 min | |
16 | 98 | 1 |
60 | 5 | |
72 | 3:20 min | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.