Team:Heidelberg/Project/Capsid Shuffling/ViroBytes
From 2010.igem.org
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*perform ViroByte Phusion HiFi Hot Start PCR to obtain desired fragments with complementary ends | *perform ViroByte Phusion HiFi Hot Start PCR to obtain desired fragments with complementary ends | ||
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**98°C 30s 1x | **98°C 30s 1x | ||
**98°C 10s | **98°C 10s | ||
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- | *digestion with Bsa1 | + | *digestion with Bsa1 - leave digesting longer than in standard protocol (at least ~3hrs) |
==='''Anchor preparation'''=== | ==='''Anchor preparation'''=== | ||
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*heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature | *heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature | ||
- | '''Anchor docking''' | + | ==='''Anchor docking'''=== |
*after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor | *after washing the beads and aspiration of the cleared solution the beads are ready for adding of the Anchor | ||
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*applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer | *applying magnet, wash twice with 80ul of wash/binding buffer (0.5 M NaCl; 20 mM Tris HCl (pH 7.5); 1 mM EDTA) and once with 80ul of 1X T4 ligase buffer | ||
- | '''BioByte-like assembly''' | + | ==='''BioByte-like assembly'''=== |
*Bead preparation | *Bead preparation |
Latest revision as of 03:57, 28 October 2010
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