Team:Heidelberg/Templates/DelH week18
From 2013.igem.org
26-08 - 01-09-13
Generation of DelH Plasmid pHM05 26-08
Overview on Fragments
Fragment | Concentration [ng/µl] | Date |
---|---|---|
G0 | 6.4 | 22-8 |
G1/2a | 8.5 | 22-08 |
G2b | 18.6 | 09-08 |
BB | 40.6 | 22-08 |
TetR | 132.1 | 26-08 |
Gibson Assembly
Mix name | DelH-G0 | DelH-1/2a | DelH-G2b | BB17 | TetR (1:10) | GibsonMasterMix | Final volume |
---|---|---|---|---|---|---|---|
0 | 8.5 µl | - | - | 0.5 µl | 1 µl | 10 µl | 20 µl |
1 | - | 6.5 µl | 2 µl | 0.5 µl | 1 µl | 10 µl | 20 µl |
- Incubate the Gibson Assembly for 1 h at 50°C in Thermocycler
Electroporation
In the next step, we purify 10 µl with Isoprop purification protocol and another 5 µl we dilute with ddH2O (10 µl)
Sample | Amount | Used in Elektroporation |
---|---|---|
0 A | 5 µl + 10 µl ddH2O |
|
0 B | 20 µl isoprop purified |
|
1 A | 5 µl + 10 µl ddH2O |
|
1 B | 20 µl isoprop purified |
|
Colony-PCR CP.W18.A
- 10 colonies of plate 1-8 (names are explained in the following table)
- PC = picked colony
- S5 = Sample 5 (watch names above on construct DelH-BB)
Template | 10 x 1 PC S5 | 50 x 1 PC S5 | 4 x 1 PC S3 | 4 x 1 PC S3 |
---|---|---|---|---|
Expected length [bp] | 663 | 663 | 663 | 663 |
Named | A -J (5 colonies per tube) | A -J (5 colonies per tube) | 1-4 | 1-4 |
Primer fw 10 µM | 10 x 1 µl VF2 | 10 x 1 µl VF2 | 4 x 1 µl VF2 | 4 x 1 µl VF2 |
Primer rev 10 µM | 10 x 1 µl DN07 | 10 x 1 1 µl DN07 | 1 µl DN07 | 1 1 µl DN07 |
Dream-Taq Polymerase (2x) | 10 x 10 µl | 10 x 10 µl | 4 x 10 µl | 4 x 10 µl |
ddH2O | 10 x 8 µl | 10 x 8 µl | 4 x 8 µl | 4 x 8 µl |
Cycles | Temperature Screening start [°C] | Time [s] |
---|---|---|
1 | 95 | 120 |
12 | 95 | 60 |
68 (touchdown -0.5°C) | 30 | |
72 | 45 | |
14 | 95 | 60 |
65 | 30 | |
72 | 45 | |
1 | 72 | 5 min |
1 | 12 | inf |
Result
Expected band: 663 bp
Colonies 15, 32, 33, 35, 39, 41, 42 and 46 show a shadow specific band at ~600 bp, slightly too low.
=> ?????
Amplification of DelH G0
Gradient-PCR Conditions G0.W18.A
Reagent | G0 | G0 | G0 | G0 |
---|---|---|---|---|
Expected length [Kb] | 18 | 18 | 18 | 18 |
Named | G0 65 | G0 66 | G0 67 | G0 68 |
Template | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 1 µl DN11 | 1 µl DN11 | 1 µl DN11 | 1 µl DN11 |
Primer rev 10 µM | 1 µl HM08 | 1 µl HM08 | 1 µl HM08 | 1 µl HM08 |
Phusion Flash Ready Mix | 10 µl | 10 µl | 10 µl | 10 µl |
ddH2O | 6 µl | 6 µl | 6 µl | 6 µl |
DMSO | 1 | 1 | 1 | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
65/66/67/68 | 5 | |
72 | 4:45 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 18 Kb
Gel shows unspecific band at 5 Kb. G0 was best amplified at 67°C.
- => Fragment was cut and gel isolated.
Re-PCR Conditions G0.W18.B
Reagent | G0 | G0 |
---|---|---|
Expected length [Kb] | 18 | 18 |
Named | G0 | G0 1 |
Template | 1 µl gel extracted G0 from 22-08 | 1 µl gel extracted G0 from 26-08 |
Primer fw 10 µM | 1 µl DN11 | 1 µl DN11 |
Primer rev 10 µM | 1 µl HM08 | 1 µl HM08 |
Phusion Flash Ready Mix | 10 µl | 10 µl |
ddH2O | 6 µl | 6 µl |
DMSO | 1 | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
65 | 5 | |
72 | 4:45 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 18 Kb
A slight band can be observed at 18 Kb, but 5Kb is much more amplified.
- => By NCBI-Blast we found out that primer HM08 can also bind inside of DelH with 6 mismatches and the forward primer DN11 can also bind with 4 mismatches inside the DelH = possible explanation for 5 Kb.
- => Change the amplification conditions.
PCR Conditions G0.W18.C
Reagent | G0 | G0 | G0 |
---|---|---|---|
Expected length [Kb] | 18 | 18 | 18 |
Named | 0 | 1 | 2 |
Template | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 1 µl DN11 | 1 µl DN11 | 1 µl DN11 |
Primer rev 10 µM | 1 µl HM08 | 1 µl HM08 | 1 µl HM08 |
Phusion II Polymerase (Hot Start) | 0.2 µl | 0.2 µl | 0.2 µl |
dNTPs | 0.4 µl | 0.4 µl | 0.4 µl |
GC buffer (5x) | 4 µl | 4 µl | 4 µl |
ddH2O | 12.4 µl | 11.4 µl | 10.4 µl |
DMSO | 0 µl | 1 µl | 2 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 30 |
30 | 98 | 10 |
65 | 20 | |
72 | 5:00 min | |
1 | 72 | 10 min |
1 | 12 | inf |
Result
Expected band: 18 Kb
Only PCR with 2 µl DMSO shows any band, but not the expected at 18 Kb, only the band at 5 Kb.
- => Further improve PCR condtions.
Amplification of DelH G1/2a
Re-PCR Conditions G1/2a.W18.A
Reagent | G1/2a |
---|---|
Expected length [Kb] | 18 |
Named | G1/2a |
Template | 1 µl gel extracted G0 from 22-08 |
Primer fw 10 µM | 1 µl DN11 |
Primer rev 10 µM | 1 µl HM06 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 6 µl |
DMSO | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
65 | 5 | |
72 | 4:45 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 18 Kb
Expected fragment was amplified, but gel shows numerous side bands.
- => Fragment was cut and gel isolated.
Gradient-PCR Conditions G1/2a.W18.A
Reagent | G1/2a | G1/2a | G1/2a | G1/2a |
---|---|---|---|---|
Expected length [Kb] | 13 | 13 | 13 | 13 |
Named | 1 | 2 | 3 | F |
Template | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl gel extracted G1/2a fragment |
Primer fw 10 µM | 1 µl DN11 | 1 µl DN11 | 1 µl DN11 | 1 µl DN11 |
Primer rev 10 µM | 1 µl HM06 | 1 µl HM06 | 1 µl HM06 | 1 µl HM06 |
Phusion Flash Ready Mix | 10 µl | 10 µl | 10 µl | 10 µl |
ddH2O | 6 µl | 5 µl | 4 µl | 6 µl |
DMSO | 1 | 2 | 3 | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
67 | 5 | |
72 | 4:45 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Lid preheated at 98°C
- No hot start
Result
- => There is no band at 18 Kb visible - entire DelH fragment could not be amplified.
Amplification of DelH FS64 - FS71
PCR Conditions FS64-FS71.W18.A
Reagent | DN11-FS66 | FS67-FS68 | FS69-HM08 | FS69-FS70 | FS71-HM08 | FS67 -HM08 |
---|---|---|---|---|---|---|
Expected length [Kb] | 4.7 | 4.0 | 9.7 | 7.0 | 2.8 | 14.0 |
Named | 1 | 2 | 3 | 4 | 5 | 6 |
Template | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl DN11 | 2.5 µl FS67 | 2.5 µl FS69 | 2.5 µl FS69 | 2.5 µl FS71 | 2.5 µl FS67 |
Primer rev 10 µM | 2.5 µl FS66 | 2.5 µl FS68 | 2.5 µl HM08 | 2.5 µl FS70 | 2.5 µl HM08 | 2.5 µl HM08 |
Phusion Flash Ready Mix | 10 µl | 10 µl | 10 µl | 10 µl | 10 µl | 10 µl |
ddH2O | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl | 3 µl |
DMSO | 1 | 1 | 1 | 1 | 1 | 1 |
Cycles | Temperature DN11-FS66 [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68↓ | 5 | |
72 | 1:25 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 85 | |
1 | 72 | 6 min |
1 | 10 | inf |
Cycles | Temperature [°C] | Time FS67 - FS68 | Time FS69-HM08 | Time FS69-FS70 | FS67 -HM08 | Time FS71-HM08 |
---|---|---|---|---|---|---|
1 | 98 | 5 | 5 | 5 | 5 | 5 |
12 | 98 | 1 | 1 | 1 | 1 | 1 |
66↓ | 5 | 5 | 5 | 5 | 5 | |
72 | 70 | 3:00 min | 2:10 min | 4:38 min | 52 | |
18 | 98 | 1 | 1 | 1 | 1 | 1 |
64 | 5 | 5 | 5 | 5 | 5 | |
72 | 70 | 3:00 min | 2:10 min | 4:38 min | 52 | |
1 | 72 | 5 min | 10 min | 5 min | 5 min | 5 min |
1 | 10 | inf | inf | inf | inf | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected bands: DN11-FS66 = 4.7 Kb, FS67-FS68 = 4.0 Kb, FS69-HM08 = 9.7 Kb, FS69-FS70 = 7.0 Kb, FS71-HM08 = 2.7 Kb, FS67-HM08 = 14 Kb
Gel shows expected fragments of DN11-FS66, FS67-FS68 and FS71-HM08
- => Bands were cut. Run gel with remaining sample for gel extraction.
- => Optimize remaining PCRs.
Amplification of DelH DN11-FS66
PCR Conditions DN11-FS66.W18.A
Reagent | 4x DN11 - FS66 |
---|---|
Expected length [Kb] | 4.7 |
Named | 1 |
Template | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl DN11 |
Primer rev 10 µM | 2.5 µl FS66 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 3 µl |
DMSO | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68↓ | 5 | |
72 | 85 | |
18 | 98 | 1 |
66 | 5 | |
72 | 85 | |
1 | 72 | 6 min |
1 | 10 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 4.7 Kb
Bands could not be used, because too much UV light was applied.
Amplification of DelH FS67-FS68
PCR Conditions FS67-FS68.W18.A
Reagent | 4x FS67-FS68 |
---|---|
Expected length [Kb] | 4.0 |
Template | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl FS67 |
Primer rev 10 µm | 2.5 µl FS68 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 3 µl |
DMSO | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
66↓ | 5 | |
72 | 1:10 min | |
18 | 98 | 1 |
64 | 5 | |
72 | 70 | |
1 | 72 | 5 min |
1 | 10 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 4.0 Kb
Bands could not be used, because too much UV light was applied.
Amplification of DelH FS71-HM08
PCR Conditions FS71-HM08.W18.A
Reagent | FS71-HM08 |
---|---|
Expected length [Kb] | 2.8 |
Template | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl FS71 |
Primer rev 10 µM | 2.5 µl HM08 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 3 µl |
DMSO | 1 |
Cycles | Temperature A [°C] | Time | Temperature B [°C] | Temperature C [°C] |
---|---|---|---|---|
1 | 98 | 5 | 98 | 98 |
12 | 98 | 1 | 98 | 98 |
68↓ | 5 | - | 69 | |
72 | 52 | 72 | 72 | |
18 | 98 | 1 | 98 | 98 |
66 | 5 | - | 69 | |
72 | 52 | 72 | 72 | |
1 | 72 | 6 min | 72 | 72 |
1 | 10 | inf | 10 | 10 |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 2.8 Kb
Bands could not be used, because too much UV light was applied.
PCR Conditions FS71-HM08.W18.B
Reagent | FS71-HM08 |
---|---|
Expected length [Kb] | 2.8 |
Template | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl FS71 |
Primer rev 10 µM | 2.5 µl HM08 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 3 µl |
DMSO | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
72.3 | 5 | |
72 | 52 | |
30 | 98 | 1 |
66 | 5 | |
72 | 52 | |
1 | 72 | 6 min |
1 | 10 | inf |
- Lid preheated at 98°C
- No hot start
Result
Expected band: 2.8 Kb
Both condistions resulted in the expected fragment.
- => Fragments were cut and gel extracted.
- => Run new PCR with 72.3°C constant annealing temperature because of the slight side bands.
Amplification of DelH FS69-FS70
PCR Conditions FS69-FS70.W18.A
Reagent | FS69-FS70 |
---|---|
Expected length [Kb] | 7.0 |
Named | FS69-FS70 |
Template | 1 µl D. acidovorans glycerol stock |
Primer fw 10 µM | 2.5 µl FS69 |
Primer rev 10 µM | 2.5 µl FS70 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 3 µl |
DMSO | 1 |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
66↓ | 5 | |
72 | 2:10 min | |
18 | 98 | 1 |
64 | 5 | |
72 | 2:10 min | |
1 | 72 | 6 min |
1 | 10 | inf |
- Lid preheated at 98°C
- No hot start
Characterization of Amplified DelH Fragments
Test Restriction Digest
Fragment | Fragment length [Kb] | Digestes with following enzymes | Expected bands [Kb] |
---|---|---|---|
DN11-FS66 | 4.7 | EcoRV | 3.7 & 0.9 |
FS67-FS68 | 4.0 | NotI | 2.7 & 1.3 |
FS69-FS70 | 7.0 | SalI | 5.5 & 1.6 |
FS71-HM08 | 2.8 | NotI & HindIII | 1.3 & 1.1 & 0.3 |
Fragment | Amount in digest | Enzyme | Buffer | ddH2O | Final amount |
---|---|---|---|---|---|
DN11-FS66 | 5 µl | 1 µl EcoRV | 2 µl 3.1 buffer | 10.9 µl | 20 µl |
FS67-FS68 | 5 µl | 1 µl NotI | 2 µl 3.1 buffer | 10.9 µl | 20 µl |
FS69-FS70 | 5 µl | 1 µl SalI-HF | 2 µl CutSmart buffer | 12 µl | 20 µl |
FS71-HM08 | 5 µl | 1 µl NotI-HF & HindIII-HF | 2 µl CutSmart buffer | 11 µl | 20 µl |
Result
Expected banda are listed above.
The test digest showed that the first fragment is cut, because the band is around 3.7 Kb, but the third and fourth lane on the gel picture show the complete non-digested fragment and on line 5 the FS71-HM08 fragment is not visible.
- => check again the digests.
Generation of DelH Plasmid pHM04 and pHM05 30-08
Gibson Assembly
Fragment | Concentration [ng/µl] | Amount with BB16 & tetR [µl] | Amount with BB17 [µl] | Amount with mRFP BB of pHM03 |
---|---|---|---|---|
DN11-FS66 | 172.5 | 1.67 | 1.47 | 1.67 |
FS67-FS68 | 120.7 | 2.03 | 1.79 | 2.03 |
FS69-FS70 | 211.3 | 2.03 | 1.79 | 2.03 |
FS71-HM08 | 146 | 1.18 | 1.04 | 1.18 |
BB16 | 40.3 | 2.54 | 0 | 0 |
tetR (1:10) | 13.2 | 0.56 | 0 | 0 |
BB17 | 23.0 | 0 | 3.91 | 0 |
BB+mRFP (pHM03) | 0 | 0 | 0 | 3.08 |
Electroporation of pHM04
10 µl H2O were added to 5 µl of the Gibson assembly mix. 1 µl and 14 µl were electroporated into electrocompetent DH10ß. Cells were incubated at 37°C for 1 h and streaked (10% vs. 90%) on LB Amp plates and stored ON at 37°C.
Colony-PCR Conditions CP.W18.B
14 colonies (#7 was left out) were picked and screened for correct integration of first DelH fragmenta s well as grown in 500 µl LB Amp.
Template | DelH construct pHM04 30-08 |
---|---|
Expected length [bp] | 663 |
Named | Colonies 1 - 15 |
Primer 10 µM fw | 2 µl VF2 |
Primer 10 µM rev | 2 µl DN07 |
Dream-Taq Polymerase (2x) | 10 µl |
ddH2O | 6 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 120 |
12 | 95 | 60 |
66 (touchdown -0.5°C) | 30 | |
72 | 2:30 min | |
18 | 95 | 60 |
65 | 30 | |
72 | 2:30 min | |
1 | 72 | 5 min |
1 | 12 | inf |
Result
Expected band: 663 bp
Colonies 1, 3, 4, 6, 12 and 15 show positive band.
- =>Of the 250 µl liquid culture, screening PCRs for the correct insertion of the last DelH fragment were performed. To the remaining 250 µl, 8 ml LB Amp were added. Colonies 1 and 3 did not grow and were excluded from further analysis.
Colony-PCR CP.W18.C
Template | pHM04 30-08 |
---|---|
Expected length [Kb] | 2.5 |
Named | Colonies 1 - 15 |
Primer 10 µM fw | 2 µl HM13 |
Primer 10 µM rev | 2 µl VR |
Dream-Taq Polymerase (2x) | 10 µl |
ddH2O | 6 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 95 | 120 |
12 | 95 | 60 |
65 (touchdown -0.5°C) | 30 | |
72 | 2:30 min | |
18 | 95 | 60 |
65 | 30 | |
72 | 2:30 min | |
1 | 12 | inf |
Results
Expected band: 2.5 Kb
Colonies 4, 6, 12 and 15 show positive band.
=> Of 2 ml of the cultures, glycerol stocks were prepared. 3 ml were mini preped and test digested. The remaining 3 ml were stored.
Test Restriction Digest of Colony S4
Clone | Concentration [ng/µl] | Amount in digest | Enzyme | Buffer | ddH2O | Final amount |
---|---|---|---|---|---|---|
pHM04 S4 | 400 ng/µL | 1 µl | 1 µl EcoRV | 1 µl CutSmart buffer | 8 µl | 10 µl |
pHM04 S4 | 400 ng/µL | 1 µl | 1 µl NotI | 1 µl CutSmart buffer | 8 µl | 10 µl |
pHM04 S4 | 400 ng/µL | 1 µl | 1 µl SalI-HF | 1 µl CutSmart buffer | 8 µl | 10 µl |
Result
Expected bands are shown in table below.
Clone | Clone length [bp] | Digestes with following enzymes | Expected bands [bp] |
---|---|---|---|
pHM04 S4 | 22,937 | EcoRI | 17,829 & 5,108 |
pHM04 S4 | 22,937 | NotI | 11,079, 6201, 3,998 & 1,628 |
pHM04 S4 | 22,937 | PvuI | 11,621, 8628 & 2,685 |
- The expected bands were not there. So the colony seems not to have the entire DelH-plasmid. .