Team:Heidelberg/Notebook/ViroBytes/October
From 2010.igem.org
(→5/10/2010) |
(→October) |
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*~200ng of fragment DNA per reaction | *~200ng of fragment DNA per reaction | ||
*ligation time ~10min | *ligation time ~10min | ||
+ | ==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 | ||
+ | |||
+ | |||
+ | [[Image:hot start 25102010 b.png|thumb|350px|center|fragments 5-8 of AAV2 and 1-8 of AAV6]] | ||
+ | |||
+ | ==26/10/2010== | ||
+ | |||
+ | * Assembly of 1-4, 5-8 and 1-8 assemblies | ||
+ | |||
+ | 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 | ||
+ | ................................................ | ||
+ | |||
+ | [[Image:assembly 26102010.png|thumb|350px|center|assembly of 1-4 5-8 1-8 and controls]] | ||
==27/09/2010== | ==27/09/2010== | ||
Revision as of 03:17, 28 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
October4/10/2010
5/10/2010
25/10/2010
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
26/10/2010
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 ................................................ File:Assembly 26102010.png assembly of 1-4 5-8 1-8 and controls 27/09/2010AAV 1 fragments 1-4; 5-8 and 1-8 (full capsid) assembly
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