Team:Heidelberg/Notebook/ViroBytes/October
From 2010.igem.org
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**Fragments 1-4; 5-8 assembly. | **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 | ||
[[Image:Gel_546.jpg|thumb|400px|right|]] | [[Image:Gel_546.jpg|thumb|400px|right|]] |
Revision as of 03:42, 28 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
October4/10/2010
5/10/2010
15/10/2010
M - DNA 1kb ladder
18/10/2010
M - DNA 1kb ladder
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
File:Hot start 25102010 a.png fragments 1-8 of AAV1 and 1-4 of AAV2
File:Hot start 25102010 c.png fragments 1-8 of AAV8 and 1-4 of AAV5 26/10/2010
27/10/2010AAV 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 ................................................
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