Team:Heidelberg/Notebook/ViroBytes/October

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**Fragments 1-4; 5-8 assembly.
**Fragments 1-4; 5-8 assembly.
 +
*ligation time 20-25min @ 16°C
 +
*60ul of beads
 +
*60ul Anchor solution (Nanodrop - 196ng/ul)
 +
*2x wash with 100ul w/b buffer
 +
*1x wash with 100ul 1x T4 ligase buffer
 +
*gentle  flicking only to dissolve the bead pellet from the wall
[[Image:Gel_546.jpg|thumb|400px|right|]]
[[Image:Gel_546.jpg|thumb|400px|right|]]

Revision as of 03:42, 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.
  • ligation time 20-25min @ 16°C
  • 60ul of beads
  • 60ul Anchor solution (Nanodrop - 196ng/ul)
  • 2x wash with 100ul w/b buffer
  • 1x wash with 100ul 1x T4 ligase buffer
  • gentle flicking only to dissolve the bead pellet from the wall
Gel 546.jpg
  • 1 - fragments 1-4 assembly.
  • 2 - fragments control.
  • 3 - assembly ligation mix

M - DNA 1kb ladder

  • 4 - fragments 5-8 assembly.
  • 5 - fragments control.
  • 6 - assembly ligation mix

18/10/2010

    • Fragments 1-4;


Gel 547.png

M - DNA 1kb ladder

  • 1 - control (fragments 1-4 from AAV1).
  • 2 - fragments 1-4 assembly
  • 3 - fragments 1-4 assembly (2nd reaction)
  • 4 - negative control

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


File:Hot start 25102010 a.png
fragments 1-8 of AAV1 and 1-4 of AAV2


fragments 5-8 of AAV2 and 1-8 of AAV6


File:Hot start 25102010 c.png
fragments 1-8 of AAV8 and 1-4 of AAV5

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

27/10/2010

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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