Team:Heidelberg/Notebook/ViroBytes/October - 2010.igem.org
Team:Heidelberg/Notebook/ViroBytes/October
From 2010.igem.org
(Difference between revisions)
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| *~200ng of fragment DNA per reaction | | *~200ng of fragment DNA per reaction |
| *ligation time ~10min | | *ligation time ~10min |
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| + | ==15/10/2010== |
| + | |
| + | Fragments 1-4; 5-8 assembly. |
| + | |
| + | |
| + | [[Image:Gel_546.jpg|thumb|600px|right|]] |
| + | |
| + | *1 - fragments 1-4 assembly. |
| + | *2 - fragments control. |
| + | *3 - assembly ligation mix |
| + | M - DNA 1kb ladder |
| + | *4 - fragments 1-4 assembly. |
| + | *5 - fragments control. |
| + | *6 - assembly ligation mix |
| + | |
| + | |
| + | {{:Team:Heidelberg/Single_Bottom}} |
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| ==25/10/2010== | | ==25/10/2010== |
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Revision as of 03:32, 28 October 2010
October
4/10/2010
- digest of 8 fragments with BsaI-HF @ 37°C for 1hr
- 6x2ul of F1-8
- 3ul NEB4 10x
- 3ul BSA 10x
- 11ul H2O
- 1ul BsaI-HF
- purification with Nucleotide Removal Kit
- NanoDrop concentration check:
Fragment 1
| Fragment 2
| Fragment 3
| Fragment 4
| Fragment 5
| Fragment 6
| Fragment 7
| Fragment 8
|
35.5 ng/ul
| 43.5 ng/ul
| 43.0 ng/ul
| 43.0 ng/ul
| 79.0 ng/ul
| 51.0 ng/ul
| 63.0 ng/ul
| 44.0 ng/ul
|
5/10/2010
- assembly started with Quick ligase kit
- ~200ng of fragment DNA per reaction
- ligation time ~10min
15/10/2010
Fragments 1-4; 5-8 assembly.
- 1 - fragments 1-4 assembly.
- 2 - fragments control.
- 3 - assembly ligation mix
M - DNA 1kb ladder
- 4 - fragments 1-4 assembly.
- 5 - fragments control.
- 6 - assembly ligation mix
|
25/10/2010
- ViroByte PCR with Phusion High-Fidelity Hot Start
PCR was set up as follows:
10 ul Phusion HF Buffer 5x
1ul of dNTP
0.5 ul of 100 um primers
2 ul of AAV template
36 ul of water
................................................
98 °C/45 s
................................................ (1x
98 °C/15 s
72 °C/30 s
................................................ (35x)
72 °C/10 min
................................................
4 °C/ hold
fragments 5-8 of AAV2 and 1-8 of AAV6
26/10/2010
- Fragment digest, Bsa1, 3hrs, purified from gel
- 7ul fragment DNA
- 3ul NEB4 10x
- 3ul BSA 10x
- 1ul Bsa1
- 16ul nuclease free water
27/09/2010
AAV 1 fragments 1-4; 5-8 and 1-8 (full capsid) assembly
Touchdown Phusion HiFi PCR was performed according to the following protocol:
................................................
98 °C/30s
................................................ (1x
98 °C/15 s
72 °C/15 s (- 1.0 °C/cycle)
72 °C/1 min
................................................ (17x)
98 °C/15 s
55 °C/30 s
72 °C/1 min
................................................ (20x)
72 °C/10 min
................................................ (1x)
4 °C/ hold
................................................
- Gel loading scheme:
- M - GeneRuler 1kb DNA ladder
- F 1-4 - fragments 1-4 assembly
- F 5-8 - fragments 5-8 assembly
- F 1-8 - fragments 1-8 assembly
- C 1-4 - fragments 1-4 control
- C 5-8 - fragments 5-8 control
- C 1-8 - fragments 1-8 control