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Team:Heidelberg/Tyrocidine week15 ms
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Revision as of 11:19, 4 October 2013
Contents |
DNA concentrations of fragments
Analysis of DNA concentration
- Concentrations obtained through quantification gel. There are 20µl of all fragments.
| fragment | concentration [ng/µl] |
|---|---|
| 3 | 2 |
| 11 | 3 |
| 13 | 7 |
| 15 | -- |
| 3 | 35 |
| 11 | 10 |
| 12 | 10 |
| 15 | 20 |
Complete List of DNA concentrations of all fragments
| fragment | concentration [ng/µl] |
|---|---|
| 1 | 10 (25.7.), 30 (28.7.) |
| 2 | 16 (25.7.), 12 (31.7.) |
| 3 | 35 (04.8.) |
| 4 | 32 (24.7.), 30 (28.7.) |
| 5 | 12 (26.7.), 14 (31.7.) |
| 6 | 8 (26.7.) (empty), 6.5 (03.8.) |
| 7 | 12 (27.7.), 18 (31.7.) |
| 8 | 8 (26.7.), 5 (03.8.) |
| 9 | 30 (28.7.) |
| 10 | 40 (28.7.) |
| 11 | 5 (31.7.), 10 (04.8.) |
| 12 | 10 (04.8.) |
| 13 | 20 (31.7.), 7 (03.8.) |
| 14 | 40 (28.7.) |
| 15 | 11 (31.7.), 20 (04.8.) |
Gibson Assembly
| fragments (10µL) + 10 µL Gibson Mix |
fragment date length
- 29.7 2500
- 29.7 2000
- 5.8. 4000
- 29.7 2500
- 29.7 3000
- 29.7 7000
- 31.7 2000
- 29.7 7000
- 29.7 2500
- 29.7 2000
- 5.8. 5000
- 5.8. 3000
- 31.7 4000
- 29.7 2000
- 5.8. 4000
Dipeptide
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 1 | 30 | 3.14 |
| 2 | 16 | 4.71 |
| 3 | 35 | 2.15 |
Tripeptide I
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 4 | 32 | 1.02 |
| 5 | 12 | 3.27 |
| 6 | 8 | 5.71 |
Tripeptide II
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 4 | 32 | 1.25 |
| 7 | 18 | 1.77 |
| 8 | 8 | 6.98 |
Tetrapeptide II
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 9 | 30 | 1.32 |
| 10 | 40 | 0.79 |
| 11 | 10 | 3.95 |
| 14 | 10 | 0.79 |
| 15 | 20 | 3.16 |
Pictures of plates
File:Plate 12 Tyc.JPG
picture of colonies taken on 06.08.2013
missing: Tetrapeptide I
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 9 | 30 | 0.77 |
| 10 | 40 | 0.46 |
| 11 | 10 | 2.31 |
| 12 | 10 | 2.77 |
| 13 | 20 | 3.69 |
overview over Gibson constructs
Excel-Document with constructs, names, DNA-concentrations and important indications
Validation of Gibson Assembly
Restriction digest of Tripetide I, II and Tetrapeptide II
File:Tyr restriction digest.png
Samples were digested with 0.5 µl NotI. Lanes 2, 8, 10, 11, 20, 25, 37, 40, 43 seem to be positive and are used for futher analysis by restriction with other enzymes
- Minipreps of colonies picked on 06.08.2013
- Restriction digest of DNA obtained by minipreps
- with NotI
- reamplifications of fragments 6 and 8 according to Protocol-page
- did not lead to any usable DNA-sample
| what | µl |
|---|---|
| DNA | 4 (~200-800ng) |
| Cutsmart Mix | 3 |
| Enzyme | 1 |
| ddH20 | ad 30 µl |
Results
Gel-Picture taken on 07.08.2013. Different peptide constructs are annotated. For exact reference to picked colonies, see: our overview over our Gibson colonies
Restriction Digest of Tri- Di and Tetrapeptides
File:Restriction disgest SphI MfeI.png
Samples were digested with 1 µl SphI (2 years over due date) (for tri- and tetrapeptides) and MfeI (for dipeptide. All lanes show unexpected lines, however, adding the fragment-sizes leads to expected plasmid size for tetrapeptide
Restriction with MfeI and NotI was performed with samples from miniprep on 07.08.2013 that appeared positive:
| what | µl |
|---|---|
| DNA | 1200ng |
| Cutsmart Mix | 2 |
| Enzyme | 1 |
| ddH20 | ad 20 µl |
Gibson Assembly was performed with fragments needed for Tetrapeptide I according to the protocol described above.
| fragment | concentration [ng/µl] | volume for gibson assembly [µL] |
|---|---|---|
| 9 | 30 | 0.77 |
| 10 | 40 | 0.46 |
| 11 | 10 | 2.31 |
| 12 | 10 | 2.77 |
| 13 | 20 | 3.69 |
Results
As the observed bands were not expected, other colonies (Tripeptide I and II) were picked for further analysis. Samples will be send to sequencing (which????xxxxxxxxx).
Proving the results of Gibson Assembly
File:20130809 restriction NotI TriI TriII.png
restriction with NotI
File:20130809 restriction SphI TriI TriII.png
restriction with SphI
- Colonies from Gibson-Assembly were picked and grown in 3 ml 2x YT medium
- Minipreps from Tripeptide-Colonies picked the day before that were grown in 2x YT medium
- Restriction digests:
- NotI (~0.8µl per sample)
- SphI (~0.5µl per sample) (enzyme 2 years over due-date)
Results
- ==> Tripeptide I shows a good band for the insert and the backbone, Tripeptide II doesn't show any positive bands
- ==> the SphI-digest was not successful - maybe because enzyme was not working
- for reference, see the overview Excel-document
- samples were sent to sequencing:
- Dipeptide 1 (lane 2) --> from plate Dipeptide 1 (colony B) (picked on 05.08.)
- Dipeptide 2 (lane 8) --> from plate Dipeptide 3 (colony B) (picked on 05.08.)
- Tripeptide I 1 (lane 10) --> from plate Tripeptide I 1 (colony A) (picked on 05.08.)
- Tripeptide I 2 (lane 11) --> from plate Tripeptide I 1 (colony B) (picked on 05.08.)
- Tripeptide II 1 (lane 20) --> from plate Tripeptide II 1 (colony B) (picked on 05.08.)
- Tripeptide II 2 (lane 25) --> from plate Tripeptide II 3 (colony A) (picked on 05.08.)
- Tetrapeptide II 1 (lane 37) --> from plate Tetrapeptide II' 1 (colony A) (picked on 05.08.)
- Tetrapeptide II 2 (lane 40) --> from plate Tetrapeptide II' 2 (colony A) (picked on 05.08.)
- Tripeptide I new 1 (lane 5 on gel image 09.08.2013) --> from plate Tripeptide I 3 (colony C) (picked on 08.08.)
- Tripeptide I new 2 (lane 6 on gel image 09.08.2013) --> from plate Tripeptide I 3 (colony D) (picked on 08.08.)
Glycerol Stock
Glycerol Stocks of all colonies that were sent to sequencing
Glycerol Stocks of newest Di-, Tri- and Tetrapeptide - colonies ->the Gibson construct overview table for reference
Restriction Digest of Tetrapeptide-I-NRPSs
- Minipreps of Tetrapeptide I colonies and new Di-, Tri- & Tetrapeptide colonies
- --> see the Gibson construct overview table for reference
File:20130810 Restriction Digest Tetrapeptide I Colony.png
restriction with Pst1 and EcoR1
File:20130810 second Restriction Digest Tetrapeptide I Colony.png
restriction with MfeI
File:Quantification gel minipreps TetI Di TriI TetII.jpg
quantification gel of minipreps of different short-peptide-NRPSs
| Probe name | Available volume |
|---|---|
| Tet I 1a | 1,3 ml |
| Tet I 1b | 1,3 ml |
| Tet I 1c | 1,3 ml |
| Tet I 2a | 1,3 ml |
| Tet I 2b | 1,3 ml |
| Tet I 2c | 1,3 ml |
| Tet I 3a | 0,845 ml |
| Tet I 3b | 0,975 ml |
| Tet I 4a | 1,3 ml |
| Tet I 4b | 0,75 ml |
| Tet I 4c | 0,845 ml |
| Tet II 2 11 | 0,750 ml |
| Tet II 2 14 | 0,975 ml |
| Tet II 3 15 | 0,750 ml |
| Tet II 2 12 | 0,975 ml |
| Di 1 | 0,975 ml |
| Tri II 1 | 0,975 ml |
| Di 2 | 1,075 ml |
| Tri II 2 | 0,975 ml |
| Di 3A | 0,975 ml |
| Tri II 3 | 0,975 ml |
| Di 3b | 0,975 ml |
Results
Restriction digest I of Tetrapeptide I with PstI and EcoR1-HF was probably positive for probe 2b,so we drove another Restriction digest with MfeI for Validation. The result was not clear, perhaps because of the extremely high DNA concentration of our Miniprep or because of the old enzyme.
We decided to sent this probe to sequencing to be sure.
Second Restriction digest with MFE 1 for validation of 2B.
