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:

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

• 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 assembly

Gel map:

• 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

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

27/10/2010

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

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

• • 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