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Team:LMU-Munich/Jump-or-die/Schedule
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(→Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz) |
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{{:Team:LMU-Munich/Templates/Page Header}} | {{:Team:LMU-Munich/Templates/Page Header}} | ||
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| + | [[image:Jumplogo.png|250px|right|ApoControl logo]] | ||
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==Aim 1: DNA reproduction+PCR== | ==Aim 1: DNA reproduction+PCR== | ||
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| - | <b>PCR4a: replication human bak with mutagenesis (Pst1) [ ]</b> | + | <b>PCR4a: replication human bak with mutagenesis (Pst1) [X]</b> |
[[Image:PCR4a.jpg]] | [[Image:PCR4a.jpg]] | ||
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| - | <b>PCR4b: replication human bak with mutagenesis (Pst1) [ ]</b> | + | <b>PCR4b: replication human bak with mutagenesis (Pst1) [X]</b> |
[[Image:PCR4b.jpg]] | [[Image:PCR4b.jpg]] | ||
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| - | <b>PCR5: joining PCR of human bak [ ]</b> | + | <b>PCR5: joining PCR of human bak [X]</b> |
[[Image:PCR5.jpg]] | [[Image:PCR5.jpg]] | ||
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| - | <b>PCR9: replication of eGFP with attP in primer [ ]</b> | + | <b>PCR9: replication of eGFP with attP in primer [X]</b> |
[[Image:PCR9.jpg]] | [[Image:PCR9.jpg]] | ||
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<b>Note: for attB two Primers ([[Team:LMU-Munich/Cut'N'survive/Schedule/Primer18| 18attBF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer19| 19attBR]]) used.</b> | <b>Note: for attB two Primers ([[Team:LMU-Munich/Cut'N'survive/Schedule/Primer18| 18attBF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer19| 19attBR]]) used.</b> | ||
| - | ==Aim 2: Inserting PCR Products in pSB1C3 and verifying | + | ==Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence == |
| - | PCR1: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| Sequence of PCR1 from sequencing]]: confirmed [ ] | + | PCR1 (BBa_K368001): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| Sequence of PCR1 from sequencing]]: confirmed [ ] |
| - | PCR5: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR5| Sequence of PCR5 from sequencing]]: confirmed [ ] | + | PCR5 (BBa_K368017): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR5| Sequence of PCR5 from sequencing]]: confirmed [ ] |
| - | PCR6: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR6| Sequence of PCR6 from sequencing]]: confirmed [ ] | + | PCR6 (BBa_K368018): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR6| Sequence of PCR6 from sequencing]]: confirmed [ ] |
| - | PCR9: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR9| | + | PCR9 (BBa_K368021): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR9| Sequence of PCR9 from sequencing]]: confirmed [ ] |
| - | PCR10: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR10| Sequence of PCR10 from sequencing]]: confirmed [ ] | + | PCR10 (BBa_K368022): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR10| Sequence of PCR10 from sequencing]]: confirmed [ ] |
==Aim 3: Assembling Biobricks== | ==Aim 3: Assembling Biobricks== | ||
We are using the [[Team:LMU-Munich/Notebook/Protocols/13_3A_Method_for_Biobrick_assembly|3A System]] to assemble Biobricks. | We are using the [[Team:LMU-Munich/Notebook/Protocols/13_3A_Method_for_Biobrick_assembly|3A System]] to assemble Biobricks. | ||
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| + | Assembly of BioBricks: | ||
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| + | * BB1 ( BBa_K368024): Assembly [ ]; prevTRE CMV + attB site | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB1| Sequence of BB1 from sequencing]]: confirmed [ ] | ||
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| + | * BB2 ( BBa_K368002): Assembly [ ]; Human Bak + SV40PA | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB2| Sequence of BB2 from sequencing]]: confirmed [ ] | ||
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| + | * BB3 ( BBa_K368003): Assembly [ ]; prevTRE CMV+ attB site + Human Bak + SV40PA | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB3| Sequence of BB3 from sequencing]]: confirmed [ ] | ||
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| + | * BB4 ( BBa_K368004): Assembly [X]; eGFP + SV40PA | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB4| Sequence of BB4 from sequencing]]: confirmed [X] | ||
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| + | * BB5 ( BBa_K368005): Assembly [ ]; PhiC31o + SV40PA | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB5| Sequence of BB5 from sequencing]]: confirmed [ ] | ||
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| + | * BB6 ( BBa_K368006): Assembly [ ]; CMV-promoter + PhiC31o + SV40PA | ||
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| + | -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB6| Sequence of BB6 from sequencing]]: confirmed [ ] | ||
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<b> Assembling Construct 1</b> | <b> Assembling Construct 1</b> | ||
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| - | [[Image: | + | [[Image: Konstruktjump1.PNG | 400pxs | Jump construct 1]] |
- Transform into HeLa cells to see if they survive. | - Transform into HeLa cells to see if they survive. | ||
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=== Construct 2 === | === Construct 2 === | ||
| - | [[Image: | + | [[Image: Konstruktjump2.PNG | 400pxs | Jump construct 2]] |
- Sequencing | - Sequencing | ||
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=== Construct 3 === | === Construct 3 === | ||
| - | [[Image: | + | [[Image: Konstruktjump3.PNG | 400pxs | Jump construct 3]] |
- Transform into HeLa cells | - Transform into HeLa cells | ||
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==Aim 5: Testing system== | ==Aim 5: Testing system== | ||
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{{:Team:LMU-Munich/Templates/Page Footer}} | {{:Team:LMU-Munich/Templates/Page Footer}} | ||
Latest revision as of 14:44, 25 October 2010
For PCR key see PCR key
Colourcode for the primers: Annealing part, mutagenised part, other sequences integrated into primer
Primer used: 1TreF 2TreR
expected product size: 492 bp
Primer used: 7BakF 8BakMutPR
expected product size: 330 bp
Primer used: 9BakMutPF 10BakR
expected product size: 376 bp
Primer used: 7BakF 10BakR
expected product size: 688 bp
Primer used: 11PAF 12PAR
expected product size: 237 bp
Primer used: 20eGFPattPF 21eGFPR
expected product size: 808 bp
Primer used: 22PC31oF 23PC31oR
expected product size: 1888 bp
Note: for attB two Primers ( 18attBF 19attBR) used.
PCR1 (BBa_K368001): Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]
PCR5 (BBa_K368017): Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]
PCR6 (BBa_K368018): Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]
PCR9 (BBa_K368021): Insertion [ ] -> Sequence of PCR9 from sequencing: confirmed [ ]
PCR10 (BBa_K368022): Insertion [ ] -> Sequence of PCR10 from sequencing: confirmed [ ]
We are using the 3A System to assemble Biobricks.
Assembly of BioBricks:
-> Sequence of BB1 from sequencing: confirmed [ ]
-> Sequence of BB2 from sequencing: confirmed [ ]
-> Sequence of BB3 from sequencing: confirmed [ ]
-> Sequence of BB4 from sequencing: confirmed [X]
-> Sequence of BB5 from sequencing: confirmed [ ]
-> Sequence of BB6 from sequencing: confirmed [ ]
Assembling Construct 2
Assembling Construct 3
- Transform into HeLa cells to see if they survive.
-> Check the leakiness of tet-on-promoter
- Induce tet-on-promoter to see, if cells die.
-> Check if construct 1 is working
- Sequencing
- Transform into HeLa cells
- Western Blot or Proteinchromatography
-> Check if PhiC31o is read off
Contents
Aim 1: DNA reproduction+PCR
PCR1: replication of the Tet-inducible CMV promotor [X]
PCR4a: replication human bak with mutagenesis (Pst1) [X]
PCR4b: replication human bak with mutagenesis (Pst1) [X]
PCR5: joining PCR of human bak [X]
PCR6: replication of the SV40-polyadenylation site [X]
PCR9: replication of eGFP with attP in primer [X]
PCR10: Replikation of PhiC31o [X]
Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence
Aim 3: Assembling Biobricks
Assembling Construct 1
Aim 4: Testing products
Construct 1
Construct 2
Construct 3
Aim 5: Testing system
