Team:Heidelberg/Notebook/ViroBytes/October

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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

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

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


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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