From 2010.igem.org

September M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 - October M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 -   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