Team:Heidelberg/Project/Capsid Shuffling/ViroBytes
From 2010.igem.org
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==Protocols== | ==Protocols== | ||
- | + | ==='''ViroByte construction'''=== | |
- | *perform ViroByte PCR to obtain desired fragments with complementary ends | + | *perform ViroByte Phusion HiFi Hot Start PCR to obtain desired fragments with complementary ends |
+ | **98°C 30s 1x | ||
+ | **98°C 10s | ||
+ | **72°C 45s 35x | ||
+ | **72°C 10min 1x | ||
+ | **4°C hold | ||
+ | |||
- | *digestion with Bsa1 | + | *digestion with Bsa1 - leave digesting longer than in standard protocol (at least ~3hrs) |
- | **'''Anchor''' | + | ==='''Anchor preparation'''=== |
+ | |||
+ | *Mix 10ul of Anchor 5 (100uM stock) with 50ul of Anchor 12689 (100ul stock); add 1.08ml of nuclease free water; mix gently | ||
+ | |||
+ | *heat on the block to 95degrees for 2 minutes and let cool slowly to room temperature | ||
+ | |||
+ | ==='''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'''=== | |
*Bead preparation | *Bead preparation |
Latest revision as of 03:57, 28 October 2010
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