Team:Heidelberg/Notebook/ViroBytes/October

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Heidelberg/Template}} {{:Team:Heidelberg/Pagetop|note_virobytes}} =October= ==4/10/2010== *digest of 8 fragments with BsaI-HF @ 37°C for 1hr **6x2ul of F1-8 **3ul NEB4 10x **3u...)
(18/10/2010)
 
(23 intermediate revisions not shown)
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{{:Team:Heidelberg/Template}}
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{{:Team:Heidelberg/Single}}
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{{:Team:Heidelberg/Pagetop|note_virobytes}}
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{{:Team:Heidelberg/Single_Pagetop|note_virobytes}}
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{{:Team:Heidelberg/Side_Top}}
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#78b41e; border: 1.5px solid #333333;"
 +
|- border="0"
 +
! colspan="7" style="background:#009be1;" | [http://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"| ||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#01.2F09.2F2010 1]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#02.2F09.2F2010 2]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#03.2F09.2F2010 3]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#04.2F09.2F2010 4]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#05.2F09.2F2010 5]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#06.2F09.2F2010 6]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#07.2F09.2F2010 7]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#08.2F09.2F2010 8]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#09.2F09.2F2010 9]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#10.2F09.2F2010 10]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#11.2F09.2F2010 11]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#12.2F09.2F2010 12]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#13.2F09.2F2010 13]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#14.2F09.2F2010 14]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#15.2F09.2F2010 15]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#16.2F09.2F2010 16]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#17.2F09.2F2010 17]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#18.2F09.2F2010 18]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#19.2F09.2F2010 19]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#20.2F09.2F2010 20]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#21.2F09.2F2010 21]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#22.2F09.2F2010 22]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#23.2F09.2F2010 23]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#24.2F09.2F2010 24]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#25.2F09.2F2010 25]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#26.2F09.2F2010 26]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#27.2F09.2F2010 27]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#28.2F09.2F2010 28]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September#29.2F09.2F2010 29]'''||'''[http://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;" | [http://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"| ||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#01.2F10.2F2010 1]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#02.2F10.2F2010 2]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#03.2F10.2F2010 3]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#04.2F10.2F2010 4]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#05.2F10.2F2010 5]'''
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#06.2F10.2F2010 6]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#07.2F10.2F2010 7]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#08.2F10.2F2010 8]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#09.2F10.2F2010 9]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#10.2F10.2F2010 10]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#11.2F10.2F2010 11]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#12.2F10.2F2010 12]'''|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#13.2F10.2F2010 13]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#14.2F10.2F2010 14]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#15.2F10.2F2010 15]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#16.2F10.2F2010 16]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#17.2F10.2F2010 17]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#18.2F10.2F2010 18]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#19.2F10.2F2010 19]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#20.2F10.2F2010 20]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#21.2F10.2F2010 21]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#22.2F10.2F2010 22]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#23.2F10.2F2010 23]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#24.2F10.2F2010 24]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#25.2F10.2F2010 25]'''
 +
|- style="background:#f2f2f2; color:#78b41e"
 +
|'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#26.2F10.2F2010 26]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#27.2F10.2F2010 27]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#28.2F10.2F2010 28]'''||'''[http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October#29.2F10.2F2010 29]'''||'''[http://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=
Line 15: Line 57:
*NanoDrop concentration check:
*NanoDrop concentration check:
<br />
<br />
-
{| class="wikitable" border="1" style="text-align: center; width: 1000px; height: 80px;"
+
{| class="wikitable" border="1" style="text-align: center; width: 620px; height: 80px;"
|-
|-
! Fragment 1
! Fragment 1
Line 43: Line 85:
*ligation time ~10min
*ligation time ~10min
-
{{:Team:Heidelberg/Pagemiddle}}
+
==15/10/2010==
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#009be1; border: 3px solid #009be1;"
+
-
|- border="0"
+
-
! colspan="7" style="background:#009be1;" | [http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/August<font color="white">August</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'''
+
-
|-
+
-
|colspan="6"| ||'''1'''
+
-
|-
+
-
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
+
-
|-
+
-
|'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
+
-
|-
+
-
|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#22/08/2010|22]]'''
+
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/August#23/08/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|24]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#26/08/2010|26]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#27/08/2010|27]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#28/08/2010|28]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#29/08/2010|29]]'''
+
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/August#30/08/2010|30]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#31/08/2010|31]]'''||colspan="5"|
+
-
|}
+
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
+
*Fragments 1-4; 5-8 assembly.
-
|- border="0"
+
**ligation time 20-25min @ 16°C
-
! colspan="7" style="background:#4e93a4;" | [http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/September<font color="#ffecba">September</font>]
+
**60ul of beads
-
|- style="background:#4e93a4; color:white"
+
**60ul Anchor solution (Nanodrop - 196ng/ul)
-
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
+
**2x wash with 100ul w/b buffer
-
|-
+
**1x wash with 100ul 1x T4 ligase buffer
-
|colspan="2"| ||'''[[Igem2010/Main/synthetic_miR_Kit/September#01/09/2010|1]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#02/09/2010|2]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#03/09/2010|3]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#04/09/2010|4]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#05/09/2010|5]]'''
+
**gentle  flicking only to dissolve the bead pellet from the wall
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#06/09/2010|6]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#07/09/2010|7]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#08/09/2010|8]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#09/09/2010|9]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#10/09/2010|10]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#11/09/2010|11]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#12/09/2010|12]]'''
+
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#13/09/2010|13]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#14/09/2010|14]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#15/09/2010|15]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#16/09/2010|16]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#17/09/2010|17]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#18/09/2010|18]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#19/09/2010|19]]'''
+
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#20/09/2010|20]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#21/09/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#22/09/2010|22]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#23/09/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#24/09/2010|24]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#25/09/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#26/09/2010|26]]'''
+
-
|-
+
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#27/09/2010|27]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#28/09/2010|28]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#29/09/2010|29]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#30/09/2010|30]]'''||colspan="3"|
+
-
|-
+
-
| colspan="7"| <span style="color:#ffffff">-</span>
+
-
|}
+
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
+
[[Image:Gel_546.jpg|thumb|400px|center|]]
-
|- border="0"
+
 
-
! colspan="7" style="background:#4e93a4;" | [http://2010.igem.org/Igem2010/Main/ViroBytes/October<font color="#ffecba">October</font>]
+
*1 - fragments 1-4 assembly.
-
|- style="background:#4e93a4; color:white"
+
*2 - fragments control.
-
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
+
*3 - assembly ligation mix
-
|-  
+
*M - DNA 1kb ladder
-
|colspan="4"| ||'''[[Igem2010/Main/synthetic_miR_Kit/October#01/10/2010|01]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#02/10/2010|02]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#03/10/2010|03]]'''
+
*4 - fragments 5-8 assembly.
-
|-
+
*5 - fragments control.
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#04/10/2010|04]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#05/10/2010|05]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#06/10/2010|06]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#07/10/2010|07]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#08/10/2010|08]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#09/10/2010|09]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#10/10/2010|10]]'''
+
*6 - assembly ligation mix
-
|-
+
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#11/10/2010|11]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#12/10/2010|12]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#13/10/2010|13]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#14/10/2010|14]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#15/10/2010|15]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#16/10/2010|16]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#17/10/2010|17]]'''
+
----
-
|-
+
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#18/10/2010|18]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#19/10/2010|19]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#20/10/2010|20]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#21/10/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#22/10/2010|22]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#23/10/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#24/10/2010|24]]'''
+
==18/10/2010==
-
|-
+
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#25/10/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#26/10/2010|26]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#27/10/2010|27]]'''||'''28'''||'''29'''||'''30'''||'''31'''
+
*Fragments 1-4 assembly
-
|-  
+
 
-
| colspan="7"| <span style="color:#ffffff">-</span>
+
 
-
|}
+
Gel map:
-
{{:Team:Heidelberg/Bottom}}
+
 
 +
*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/Single_Bottom}}

Latest revision as of 03:57, 28 October 2010

October

4/10/2010

  • digest of 8 fragments with BsaI-HF @ 37°C for 1hr
    • 6x2ul of F1-8
    • 3ul NEB4 10x
    • 3ul BSA 10x
    • 11ul H2O
    • 1ul BsaI-HF
  • purification with Nucleotide Removal Kit
  • NanoDrop concentration check:


Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5 Fragment 6 Fragment 7 Fragment 8
35.5 ng/ul 43.5 ng/ul 43.0 ng/ul 43.0 ng/ul 79.0 ng/ul 51.0 ng/ul 63.0 ng/ul 44.0 ng/ul


5/10/2010

  • assembly started with Quick ligase kit
  • ~200ng of fragment DNA per reaction
  • 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
Gel 546.jpg
  • 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


Gel 547.png

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



fragments 5-8 of AAV2 and 1-8 of AAV6


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

................................................


Full assembly 27.10.10.jpg
    • 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