Team:Heidelberg/Templates/DelH week9
From 2013.igem.org
Contents |
24-06 - 30-06-13
Characterization of DelH plasmid 19-06
SDS Page
- Using NuPAGE Novex 10% Bis-Tris precast gel from Invitrogen with MOPS running buffer
- 3NuPAGE Novex 3-8% Tris-Acetate precast gel would have been more suitable considering expected 600 kDa DelH, but needed Tris-Acetate SDS buffer was not available.
- Resuspended pellets in 100 µl SDS buffer (from Linda)
- Boiled for 10 min @98°C
- Ran for 90 min, 180 V
- Stained for 40 min in Coomassie Buffer (50 ml acetic acid 100% + 225 ml ddH2O, 1.5 g Coomassie in 225 ml methanol, mix both)
- Washed in ddH2O multiple times
Result
None clear band at ~600 kDa visible. Maybe in highest amounts, but not reliable.
- => Probably no DelH expression in analyzed colonies.
Mini Prep
- Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9)
- Incubation ON at 37°C
- 5 ml ON cultures were mini prepared and resuspended in 50 µl
Test Restriction Digest
- 5 µl of the mini prep were test digested
Reagent | Volume [µl] |
---|---|
Miniprep DNA | 5 |
CutSmart buffer | 2 |
EcoRI-HF, PacI, KpnI-HF | 0.5 each |
ddH2O | 10.75 |
- Incubation at 37°C for 1 h
Result
Expected band pattern: 5 kp, 7.3 kp, 13 kp
No clear bands visible.
- => DNA yield from mini prep was probably too low.
DNA Concentration
Colony | DNA concentration [ng/µl] |
---|---|
1 | 124.5 |
4 | 3.9 |
6 | 1.6 |
9 | 10.2 |
DNA measurement confirmed suspicion from test digest.
- =>Thus, mini prep and test digest have to be repeated
Repetion of Mini Prep
- Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9) or from remaining ON culture (colony 1)
- Incubation ON at 37°C
- Colony 1 did not grow (also not on LB Amp plate), obviously lost plasmid
- Mini preped remaining 3, eluted in 35 µl ddH2O
DNA Concentration
Colony | DNA concentration [ng/µl] |
---|---|
4 | 38.5 |
6 | 42.0 |
9 | 49.0 |
Test Restriction Digest
- Digested entire mini prep (using remaining from first mini for colony 1) for 2 h at37°C
Reagent | Volume [µl] |
---|---|
Mini prep | 42 |
CutSmart | 5 |
EcoRI-HF, PacI, KpnI-HF | 1 each |
Result
Expected bands: BB (7.3 Kb), DelH F2 (8 Kb), F1a + F1b (10 Kb)
Unexpected bands at 3 Kb, but not desired ones.
- => Colonies do not contain desired plasmid and entire ligation has to be repeated.
Amplification of DelH F1b
PCR Conditions F1b.W9.A
Reagent | DelH F1b |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 |
Primer fw 10 µM | 0.5 µl DelH_EcoRI_fw 100 µM |
Primer rev 10 µM | 0.5 µl DelH_f1_SalI_rev 100 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
35 | 98 | 15 s |
68 | 5 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 5 Kb, Loaded 1 µl of PCR
No correct band visible.
- => Why could we not reproduce the previous amplification?
PCR Conditions F1b.W9.B
Reagent | DelH F1b |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 |
Primer fw 10 µM | 0.5 µl DelH_EcoRI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f1_SalI_rev 10 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
30 | 98 | 15 s |
68 | 15 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 5 Kb, Loaded 1 µl of PCR
Again, expected band not there.
- => Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.
PCR Conditions F1b.W9.C
Reagent | DelH F1b |
---|---|
Template | 1 µl DelH F1b Fragment |
Primer fw 10 µM | 0.5 µl DelH_EcoRI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f1_SalI_rev 10 µM |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
30 | 98 | 1 s |
68 | 5 s | |
72 | 120 | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 5 Kb, Loaded 1 µl of PCR
Highly specific band at 5 Kb, as well as additional smaller ones.
- => Band was cut. Run gel with remaining sample for gel extraction.
PCR Conditions F1b.W9.D
Reagent | DelH F1b |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 1:10 |
Primer fw 10 µM | 0.5 µl DelH_EcoRI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f1_SalI_rev 10 µM |
Q5 Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
30 | 98 | 1 s |
68 | 5 s | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: DelH F1b 5 Kb
Neither of the PCRs yield any product.
- => Therefore, Q5 is no option for us!
Amplification of DelH F2
PCR Conditions F2.W9.A
Reagent | DelH 2 |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 |
Primer fw 10 µM | 0.5 µl DelH_f2_SalI_fw 100 µM |
Primer rev 10 µM | 0.5 µl DelH_f2_KpnI_rev 100 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
35 | 98 | 15 s |
66 | 5 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 8 Kb
Unexpected bands at 3 Kb in lanes of DelH 2
- => Why could we not reproduce the previous amplification?
PCR Conditions F2.W9.B
Reagent | DelH 2 |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 |
Primer fw 10 µM | 0.5 µl DelH_f2_SalI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f2_KpnI_rev 10 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
12 | 98 | 15 s |
66 decrease by 0.5 | 15 s | |
72 | 3:30 min | |
18 | 98 | 15 s |
66 | 15 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 8 Kb
Unexpected bands at 1.5 Kb, 2.2 Kb, 4 Kb and 6 Kb. Desired 8 Kb band is present but hardly visible.
- => Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.
PCR Conditions F2.W9.C
Reagent | DelH 2 |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 1:10 |
Primer fw 10 µM | 0.5 µl DelH_f2_SalI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f2_KpnI_rev 10 µM |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
12 | 98 | 1 s |
66 decrease by 0.5 | 5 s | |
72 | 2:30 min | |
18 | 98 | 1 s |
66 | 5 s | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 8 Kb
F2 shows specific band at 8 Kb and additional ones.
- => Band was cut. Run gel with remaining sample for gel extraction.
PCR Conditions F2.W9.D
Reagent | DelH 2 |
---|---|
Template | 1 µl D. acidovorans stock 29-04-13 1:10 |
Primer fw 10 µM | 0.5 µl DelH_f2_SalI_fw 10 µM |
Primer rev 10 µM | 0.5 µl DelH_f2_KpnI_rev 10 µM |
Q5 Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
12 | 98 | 1 s |
66 decrease by 0.5 | 5 s | |
72 | 2:30 min | |
18 | 98 | 1 s |
66 | 5 s | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 8 Kb, Loaded 1 µl of PCR
Neither of the PCRs yield any product.
- => Therefore, Q5 is no option for us!
Amplification of Backbone pSB6A1-AraC-lacZ
PCR Conditions BB.W9.A
Reagent | Backbone |
---|---|
Template | 1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl) |
Primer fw 10 µM | 0.5 µl AraCbb_KpnI_fw 100 µM |
Primer rev 10 µM | 0.5 µl AraCbbPacI_rev2 100 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
35 | 98 | 15 s |
66 | 5 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 7.3 Kb, Loaded 1 µl of PCR
No band visible, amplification failed.
- => Why could we not reproduce the previous amplification?
PCR Conditions BB.W9.B
Reagent | BB |
---|---|
Template | 1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl) |
Primer fw 10 µM | 0.5 µl AraCbb_KpnI_fw 10 µM |
Primer rev 10 µM | 0.5 µl AraCbbPacI_rev2 10 µM |
Phusion Ready Mix | 25 µl |
ddH2O | 23 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 30 s |
12 | 98 | 15 s |
66 decrease by 0.5 | 15 s | |
72 | 3:30 min | |
18 | 98 | 15 s |
66 | 15 s | |
72 | 3:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 7.3 Kb, Loaded 1 µl of PCR
Unexpected band at 2.2 Kb
- => Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.
PCR Conditions BB.W9.C
Reagent | DelH 2 |
---|---|
Template | 1 µl pSB6A1-AraC-LacZ digested and purified 1:10 |
Primer fw 10 µM | 0.5 µl AraCbb_KpnI_fw 10 µM |
Primer rev 10 µM | 0.5 µl AraCbbPacI_rev2 10 µM |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
12 | 98 | 1 s |
66 decrease by 0.5 | 5 s | |
72 | 2:30 min | |
18 | 98 | 1 s |
66 | 5 s | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 7.3 Kb, Loaded 1 µl of PCR
Gel does not show expected fragment.
- => Make sure right template was used. Is the restricted and purified fragment suitable?
PCR Conditions BB.W9.D
Reagent | DelH 2 |
---|---|
Template | 1 µl pSB6A1-AraC-LacZ digested and purified 1:10 |
Primer fw 10 µM | 0.5 µl AraCbb_KpnI_fw 10 µM |
Primer rev 10 µM | 0.5 µl AraCbbPacI_rev2 10 µM |
Q5 Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 10 s |
12 | 98 | 1 s |
66 decrease by 0.5 | 5 s | |
72 | 2:30 min | |
18 | 98 | 1 s |
66 | 5 s | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Using hot start at 98°C
Result
Expected band: 7.3 Kb, Loaded 1 µl of PCR
Neither of the PCRs yield any product.
- => Therefore, Q5 is no option for us!