Team:Heidelberg/Notebook/ViroBytes/October

From 2010.igem.org

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*~200ng of fragment DNA per reaction
*~200ng of fragment DNA per reaction
*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/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


Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/ViroBytes/October"