Team:Heidelberg/Templates/DelH week11
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08-07 - 14-07-13
Amplification of DelH F1b
PCR Conditions F1b.W11.A
Reagent | DelH F1b - from purified F1b 05-07 (genome amplified) | DelH F1b - from purified F1b 05-07 (genome amplified) |
---|---|---|
Template | 1 µl of PCR 1b (19 ng/µl) | 1 µl D. acidovorans |
Primer fw 10 µM | 1 µl DelH_EcoRI_fw | 1 µl DelH_EcoRI_fw |
Primer rev 10 µM | 1 µl DelH_f1_SalI_rev 10 | 1 µl DelH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl | 10 µl |
ddH2O | 7 µl | 6 µl |
DMSO | - | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
68 | 5 | |
72 | 2:15 min | |
1 | 72 | 7 min |
1 | 4 | inf |
Result
Expected band: 5 Kb
There is a specific band, but not at 5 Kb.
- => We cannot amplify F1b from a PCR fragment. Use fresh bacteria instead.
Preparation of fresh ACM media
- Mix ingrediants according to Acidovorax complex medium
- Autoclave
- pH adjust to 7,3
- Mix 500 ml media and 6 g agar
- Autoclave again
- Pour plates
Preparation of fresh D. acidovorans
- Inocculate 5 ml ACM media with D. acidovorans
- Incubate ON
- Prepare glycerol stock
- Streak some of culture on plates
PCR Conditions F1b.W11.B
Reagent | DelH F1b | DelH F1b |
---|---|---|
Template | Fresh colony of plate by DN from 10-07 | Fresh colony of plate by DN from 10-07 |
Primer fw 10 µM | 1 µl delH_EcoRI_fw | 1 µl delH_EcoRI_fw |
Primer rev 10 µM | 1 µl delH_f1_SalI_rev | 1 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl | 10 µl |
ddH2O | 7 µl | 6 µl |
DMSO | - | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0.5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5.4 Kb
There is a weak band at the probe 1b WITH DMSO above 5 Kb.
- => Start a new PCR with 50 µl with the same conditions.
PCR Conditions F1b.W11.C
Reagent | DelH 1b |
---|---|
Template | Fresh colony of plate by DN from 10-07 |
Primer fw 10 µM | 2.5 µl delH_EcoRI_fw |
Primer rev 10 µM | 2.5 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 25 µl |
ddH2O | 17.5 µl |
DMSO | 2.5 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0.5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5 Kb.
The expected band at 5 Bk is weak, but visible. Note that there is sample left in the pocket.
- => The fragment was cut and gel extracted.
Generation of DelH plasmid
Restriction Digest
Of fragments F1a, F1b, F2 and backbone.
Fragment | DNA [µl] | ddH2O [µl] | Enzymes [µl] | BSA [µl] | Buffer 4 [µl] |
---|---|---|---|---|---|
F1a (1) | 30 | 7 | 1.5 Pac & EcoRI-HF each | 5 | 5 |
F1a (2) | 30 | 7 | 1.5 Pac & EcoRI-HF each | 5 | 5 |
F1b (11.7 - 20 µl) | 20 | 17 | 1.5 SalI-HF & EcoRI-HF each | 5 | 5 |
F1b (11.7 - 50 µl) | 20 | 17 | 1.5 SalI-HF & EcoRI-HF each | 5 | 5 |
F2 | 20 | 17 | 1.5 SalI-HF & KpnI-HF each | 5 | 5 |
BB (6-7 of IK - 14 ng/µl) | 20 | 17 | 1.5PacI & KpnI-HF each | 5 | 5 |
BB (6-7) | 20 | 17 | 1.5 PacI & KpnI-HF each | 5 | 5 |
Result
Loaded 5 µl of restriction digested fragments on gel. The result stands in the table below:
Fragment | Band | Conclusion | Next step |
---|---|---|---|
F1a purified on 12-07 | strong bands at 1, 2 and 3 Kb weak band at wanted 5 KB | PCR purification of F1a step was not the right purifying step | load complete probe on gel and gel extract |
F1b purified on 12-07 | weak band at 5 Kb | maybe only low concentration | run PCR again |
F2 | no band | maybe too low concentration | run PCR again |
BB | weak band at 7.4 Kb | maybe only low concentration | run PCR again |
Gel Extraction of DelH F1a
Expected band: 5 KB, loaded entire F1a purified on 12-07 on gel
No band visible.
- => Gel extract earlier PCR from 06-07.
Expected band: 5 Kb
PCR shows expected band.
- => F1a was cut and gel extracted.
Amplification of DelH F1b
PCR Conditions F1b.W11.D
Reagent | DelH F1b |
---|---|
Template | 1 µl F1b digested 12-07 |
Primer fw 10 µM | 1 µl delH_EcoRI_fw |
Primer rev 10 µM | 1 µl delH_f1_SalI_rev |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 6µl |
DMSO | 1 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
68 (touchdown -0,5°C) | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 5.4 Kb
PCR did not yield the specific band.
- => Use altered PCR conditions.
Amplification of DelH F2
PCR Conditions F2.W11.A
Reagent | DelH F2 |
---|---|
Template | 1 µl F2 digested 12-07 |
Primer fw 10 µM | 1 µl delH_f2_SalI_fw |
Primer rev 10 µM | 1 µl delH_f2_KpnI_rev |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time |
---|---|---|
1 | 98 | 5 s |
30 | 98 | 1 s |
66 | 5 s | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 8 Kb
PCR does not show the specific band.
- => Further optimization.
Amplification of Backbone
PCR Conditions BB.W11.A
Reagent | Backbone |
---|---|
Template | 1 µl backbone digested 12-07 |
Primer fw 10 µM | 1 µl AraCbb_KpnI_fw |
Primer rev 10 µM | 1 µl AraCbb_PacI_rev2 10 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
30 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Result
Expected band: 8 Kb
PCR does not show the specific band.
- => Repeat using fresh backbone DNA from miniprep.
Miniprep
- Inocculation of 10 ml LB Amp with DH10ß (pSB6A1-AraC-lacZ)
- Perform mini prep
PCR Conditions BB.W11.B
Reagent | Backbone |
---|---|
Template | 1 µl Miniprep A of 10-07 |
Primer fw 10 µM | 1 µl AraCbb_KpnI_fw |
Primer rev 10 µM | 1 µl AraCbb_PacI_rev2 |
Phusion Flash Ready Mix | 10 µl |
ddH2O | 7 µl |
Cycles | Temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 5 |
12 | 98 | 1 |
66 (touchdown -0.5°C) | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results
Expected band: 7.4 Kb, loaded miniprep and PCR product.
Miniprep shows weak band at ~7.4 Kb. PCR does not show expected band.
- => Prepare new mediprep of backbone (pSB6A1-AraC-lacZ)
Gibson Assembly Strategy
Identifier | Order date | Note | Sequence |
---|---|---|---|
DN01:DelH_f1_PacI_fw | 03-05-2013 | Amplification of DelH F1, with RBS and adding PacI restriction site | TTTT TTAATTAA TCACACAGGAAAGTACTAG ATGGACCGTGGCCGCCTGC GCCAAATCG |
DN02:DelH_f1_SalI_rev | 03-05-2013 | Amplification of DelH F1 until SalI restriction site | TTTT GTCGACCAACACCTGTGCCTGC |
DN03:DelH_f2_SalI_fw | 03-05-2013 | Amplification of DelH F2 starting at SalI restriction site | TTTT GTCGACTGGATGGAGCCTGGTGAAAG |
DN04:DelH_f2_KpnI_rev | 03-05-2013 | Amplification of DelH F2, adding KpnII restriction site | TTTT GGTACC TCAGTCCAGCGCGTACTCCAG |
DN05:AraCbb_KpnI_fw | 03-05-2013 | Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding KpnI site | TTTT GGTACC AAAAGAGGAGAAAtactagatgaccatg |
DN08:AraCbb_PacI_rev | 03-05-2013 | Amplification of backbone for DelH (pSB6A1-AraC-lacZ), adding PacI site | tttt ttaattaa gctagcccaaaaaaacgggtatg |