Team:Heidelberg/Templates/Del week15 OP
From 2013.igem.org
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(→Concentration measurement OP (07-08-2013)) |
(→Amplification II of Del OP (FS_22 to FS_13; xx kb)) |
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* PCR will be repeated at a lower annealing temperature of 55°C in case primers were not able to bind at high temperatures | * PCR will be repeated at a lower annealing temperature of 55°C in case primers were not able to bind at high temperatures | ||
- | ===Amplification II | + | ===Amplification II from FS_22 to FS_13; xx kb=== |
[[File:Heidelberg_20130810 2log 6xDelOP(55°C-const)fromgylcerolstock.png|150px|thumb|Amplifcation of DelOP (II), from glycerol stocks at 55°C const. ; run at 100 V, 0.8 % gel (TAE)(09.08)]] | [[File:Heidelberg_20130810 2log 6xDelOP(55°C-const)fromgylcerolstock.png|150px|thumb|Amplifcation of DelOP (II), from glycerol stocks at 55°C const. ; run at 100 V, 0.8 % gel (TAE)(09.08)]] | ||
Revision as of 19:07, 3 October 2013
Contents |
08-08-2013
Amplification from FS_22 to FS_13, 2.7 kb
- Reaktion
what | µl |
---|---|
D. acidovorans SPH-1 (colony-pcr) | 1 |
FS_22: (1/10) | 4 |
FS_13long: (1/10) | 4 |
Phusion flash Master Mix | 10 |
- Conditions
Biorad MyCycler* | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
50 | 5 | |
72 | 1:00 | |
1 | 72 | 5 min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated at a higher annealing temperature of 65°C to increase primer binding
Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 07-08-2013) with EcoRI-HF
Incubation at Room temperature for about 4h and at 37°C for 1 hours
what | µl |
---|---|
SR_22 to FS_13 (07-08-2013) | 20 |
EcoRI-HF | 1 |
Buffer CutSmart | 3 |
dd H2O | 6 |
Expected fragment lengths [bp] | 1883, 960 |
Results:
- Restriction digest did not work, amplicon was not cut and total amount of DNA was probably too low
- Restriction digest will be repeated as soon as PCR of DelOP works reliable and reproducable with higher amounts of DNA
Concentration measurement OP (07-08-2013)
Fragment | Primer | Date PCR | Concentration |
---|---|---|---|
DelOP | FS22-FS13 | 07-08-2013 | 6.1 ng/µl |
09-08-2013
Amplification I from FS_22 to FS_13, 2.7 kb
- Reaktion
what | µl |
---|---|
D. acidovorans SPH-1 (colony-pcr) | 1 |
FS_22: (1/10) | 4 |
FS_13long: (1/10) | 4 |
Phusion flash Master Mix | 10 |
- Conditions
Biorad T100 | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
65 | 5 | |
72 | 1:00 | |
1 | 72 | 5 min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated at a lower annealing temperature of 55°C in case primers were not able to bind at high temperatures
Amplification II from FS_22 to FS_13; xx kb
wrong Primers were used
- Reaktion
what | µl |
---|---|
D. acidovorans SPH-1 (colony-pcr) | 1 |
FS_22: (1/10) | 4 |
FS_13long: (1/10) | 4 |
Phusion flash Master Mix | 10 |
- Conditions
Biorad T100 | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
55 | 5 | |
72 | 1:00 | |
1 | 72 | 5 min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work as the wrong primers were used
- Amplification will be repeated with different amounts of DMSO
Amplification III from FS_22 to FS_13; 2.7 kb
3 samples, one with, two without DMSO (one with much template, one with less template)
- Reaktion
what | µl |
---|---|
D. acidovorans SPH-1 colony | 1 |
FS_22: (1/10) | 4 |
FS_13long: (1/10) | 4 |
Phusion flash Master Mix | 10 |
DMSO | 0/1 |
- Conditions
Biometra TProfessional Basic | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
55 | 5 | |
72 | 1:00 | |
1 | 72 | 5 min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work, neither with lower amount of template, nor with higher, furthermore the DMSO concentration did not influence the PCR for the given annealing temperature
- PCR will be repeated with recently ordered new Primers which bind outside the intended template in the Del-Cluster of D. acidovorans SPH-1 in order to obtain a specific template suited for a nested PCR of DelOP with Primers FS_22 and FS_14l
10-08-2013
Amplification I, nested PCR different Primers; 2.7 kb
- Reaction
Reagent | DelOP | ||||
---|---|---|---|---|---|
Template | D.acidovorans SPH-1 colony | ||||
Primer fw | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 |
Primer rev | 4 µl FS_13l | 4 µl FS_27 | 4 µl FS_28 | 4 µl FS_29 | 4 µl FS_30 |
Phusion Ready Mix | 10 µl | ||||
dd H2O | 1 µl |
- Conditions
Biometra TProfessional Basic | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
55 | 5 | |
72 | 1:00 | |
1 | 72 | 5 min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated as touchdown PCR with annealing starting at 68°C and different primer combinations to the template needed for the nested PCR of DelOP with primers FS_22 to FS_13l
Amplification II, nested PCR with different primers; 2.7 kb
- Reaction
Reagent | DelOP | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Template | D.acidovorans SPH-1 colony | |||||||||
Primer fw | 4 µl FS_22 | 4 µl FS_22 | 4 µl FS_33 | 4 µl FS_33 | 4 µl FS_31 | 4 µl FS_31 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_22 | 4 µl FS_22 |
Primer rev | 4 µl FS_13s | 4 µl FS_27 | 4 µl FS_13s | 4 µl FS_27 | 4 µl FS_29 | 4 µl FS_30 | 4 µl FS_29 | 4 µl FS_30 | 4 µl FS_28 | 4 µl FS_29 |
DMSO | 1 µl | |||||||||
Phusion Ready Mix | 10 µl | |||||||||
dd H2O | 1 µl |
- Conditions
Biometra TProfessional Basic | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 1:00 | |
18 | 98 | 1 |
65 | 5 | |
72 | 1:00 | |
1 | 72 | 10min |
1 | 12 | inf |
Results:
- Amplification of DelOP worked with the Primer combinations FS22-FS13short, FS_22-FS_27, FS_33-FS_13short, FS_33-FS_27, FS_32-FS_30 and FS_22-FS28
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- all the obtained PCR products will be used as specific templates for a nested PCR using primers FS_22-FS_13long
Amplification III from FS_22 to FS_13; 2.7 kb
- Reaction
Reagent | DelOP | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Template | D.acidovorans SPH-1 colony | |||||||||
Primer fw | 4 µl FS_22 | |||||||||
Primer rev | 4 µl FS_13l | |||||||||
DMSO | 1 µl | |||||||||
Phusion Ready Mix | 10 µl | |||||||||
dd H2O | 1 µl |
- Conditions
Biometra TProfessional Basic | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 1:00 | |
18 | 98 | 1 |
65 | 5 | |
72 | 1:00 | |
1 | 72 | 10min |
1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- hope and wait for the nested PCR
11-08-2013
Nested PCR of DelOP-Fragments from 10-08-2013
- Reaction
Reagent | DelOP | ||||||
---|---|---|---|---|---|---|---|
Template | FS22-13s | FS31-30 | FS33-27 | FS22-28 | FS22-13s | FS31-30 | FS33-29 |
Primer fw | 4.5 µl FS_22 | ||||||
Primer rev | 4.5 µl FS_13l | ||||||
DMSO | 1 µl | ||||||
Phusion Ready Mix | 10 µl |
- Conditions
Biometra TProfessional Basic | |||||
---|---|---|---|---|---|
Cycles | temperature [°C] Template FS22-13s, FS31-30, FS33-27, FS22-28 | Time | Cycles | temperature [°C] Template FS22-13s, FS31-30, FS33-29, Time | |
1 | 98 | 10 s | 1 | 98 | 10 s |
30 | 98 | 1 s | 12 | 98 | 1 s |
68 ↓ 0.5 | 5 s | ||||
58 | 5 s | 72 | 1:00 min | ||
18 | 98 | 1 s | |||
72 | 1:00 min | 66 | 5 s | ||
72 | 1:00 min | ||||
1 | 72 | 5 min | 1 | 72 | 5 min |
1 | 10 | inf | 1 | 10 | inf |
Results:
- Nested PCR of DelOP did not work, though concentrations of the used template were fine as checked previously
- PCR will be with an annealing temperature of 55°C with the template obtained from the primer combination of FS_22-FS_13short as nested PCR