Team:Heidelberg/Notebook/ViroBytes/October
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- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single}} |
- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single_Pagetop|note_virobytes}} |
+ | {{:Team:Heidelberg/Side_Top}} | ||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#78b41e; border: 1.5px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September<font color="#ffecba">September</font>] | ||
+ | |- style="background:#009be1; color:white" | ||
+ | |width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |colspan="2"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#01.2F09.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#02.2F09.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#03.2F09.2F2010 3]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#04.2F09.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#05.2F09.2F2010 5]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#06.2F09.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#07.2F09.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#08.2F09.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#09.2F09.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#10.2F09.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#11.2F09.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#12.2F09.2F2010 12]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#13.2F09.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#14.2F09.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#15.2F09.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#16.2F09.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#17.2F09.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#18.2F09.2F2010 18]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#19.2F09.2F2010 19]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#20.2F09.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#21.2F09.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#22.2F09.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#23.2F09.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#24.2F09.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#25.2F09.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#26.2F09.2F2010 26]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#27.2F09.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#28.2F09.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#29.2F09.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#22.2F30.2F2010 30]'''||colspan="3"| | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | | colspan="7"| <span style="color:#ffffff">-</span> | ||
+ | |} | ||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#78b41e;" | [https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October<font color="white">October</font>] | ||
+ | |- style="background:#78b41e; color:white" | ||
+ | |width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#03.2F10.2F2010 3]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#05.2F10.2F2010 5]''' | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#10.2F10.2F2010 10]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#12.2F10.2F2010 12]'''|'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#15.2F10.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#17.2F10.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#18.2F10.2F2010 18]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#19.2F10.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#20.2F10.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#21.2F10.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#22.2F10.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#23.2F10.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#24.2F10.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#25.2F10.2F2010 25]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#26.2F10.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#27.2F10.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#28.2F10.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#29.2F10.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#22.2F30.2F2010 30]'''||colspan="3"| | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |colspan="7"| | ||
+ | <span style="color:#ffffff">-</span> | ||
+ | |} | ||
+ | {{:Team:Heidelberg/Side_Bottom}} | ||
+ | __NOTOC__ | ||
=October= | =October= | ||
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*ligation time ~10min | *ligation time ~10min | ||
- | + | ==15/10/2010== | |
- | + | *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|center|]] | |
- | + | ||
- | + | *1 - fragments 1-4 assembly. | |
- | + | *2 - fragments control. | |
- | + | *3 - assembly ligation mix | |
- | + | *M - DNA 1kb ladder | |
- | + | *4 - fragments 5-8 assembly. | |
- | | | + | *5 - fragments control. |
- | + | *6 - assembly ligation mix | |
- | + | ||
+ | ---- | ||
+ | |||
+ | ==18/10/2010== | ||
+ | |||
+ | *Fragments 1-4 assembly | ||
+ | |||
+ | |||
+ | Gel map: | ||
+ | |||
+ | *M - DNA 1kb ladder | ||
+ | *1 - control (fragments 1-4 from AAV1). | ||
+ | *2 - fragments 1-4 assembly | ||
+ | *3 - fragments 1-4 assembly (2nd reaction) | ||
+ | *4 - negative control | ||
+ | |||
+ | |||
+ | [[Image:Gel_547.png|thumb|500px|center|]] | ||
+ | |||
+ | ---- | ||
+ | |||
+ | ==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]] | ||
+ | |||
+ | <br /> | ||
+ | |||
+ | ==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 | ||
+ | |||
+ | ==27/10/2010== | ||
+ | |||
+ | 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 | ||
+ | |||
+ | ................................................ | ||
+ | |||
+ | |||
+ | [[Image:Full_assembly_27.10.10.jpg|thumb|600px|right|]] | ||
+ | |||
+ | **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 | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
+ | {{:Team:Heidelberg/Single_Bottom}} | ||
- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single_Bottom}} |
Latest revision as of 03:57, 28 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
October4/10/2010
5/10/2010
15/10/2010
18/10/2010
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
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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