Team:Heidelberg/Notebook/ViroBytes/October

From 2010.igem.org

(Difference between revisions)
(→5/10/2010)
(→October)
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*~200ng of fragment DNA per reaction
*~200ng of fragment DNA per reaction
*ligation time ~10min
*ligation time ~10min
 +
==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
 +
 +
 +
[[Image:hot start 25102010 b.png|thumb|350px|center|fragments 5-8 of AAV2 and 1-8 of AAV6]]
 +
 +
==26/10/2010==
 +
 +
* Assembly of 1-4, 5-8 and 1-8 assemblies
 +
 +
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
 +
................................................
 +
 +
[[Image:assembly 26102010.png|thumb|350px|center|assembly of 1-4 5-8 1-8 and controls]]
==27/09/2010==
==27/09/2010==

Revision as of 03:17, 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

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

  • Assembly of 1-4, 5-8 and 1-8 assemblies

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 ................................................

File:Assembly 26102010.png
assembly of 1-4 5-8 1-8 and controls

27/09/2010

AAV 1 fragments 1-4; 5-8 and 1-8 (full capsid) assembly

Full assembly 27.10.10.jpg


    • 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

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