Team:LMU-Munich/Cut'N'survive/Schedule
From 2010.igem.org
(→Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz) |
|||
Line 86: | Line 86: | ||
==Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz == | ==Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz == | ||
- | PCR1: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| | + | PCR1: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| Sequence of PCR1 from sequencing]]: confirmed [ ] |
- | PCR3: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR3| | + | PCR3: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR3| Sequence of PCR3 from sequencing]]: confirmed [ ] |
- | PCR5: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR5| | + | PCR5: 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| | + | PCR6: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR6| Sequence of PCR6 from sequencing]]: confirmed [ ] |
- | PCR8: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR8| | + | PCR8: Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR8| Sequence of PCR8 from sequencing]]: confirmed [ ] |
==Aim 3: Assembling Biobricks== | ==Aim 3: Assembling Biobricks== |
Revision as of 10:38, 21 September 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: 492bp
Primer used: 3TevF 4TevMutEPR
expected product size: 332 bp
Primer used: 5TevMutEPF 6TevR
expected product size: 772 bp
Primer used: 3TevF 6TevR
expected product size: 1087 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: 13TrecF 14TrecMutSR
expected product size: 850 bp
Primer used: 15TrecMutSF 16TrecR
expected product size: 402 bp
Primer used: 13TrecF 16TrecR
expected product size: 1233 bp
PCR1: Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]
PCR3: Insertion [ ] -> Sequence of PCR3 from sequencing: confirmed [ ]
PCR5: Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]
PCR6: Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]
PCR8: Insertion [ ] -> Sequence of PCR8 from sequencing: confirmed [ ]
We are using the 3A System to assemble Biobricks.
Assembling Construct 1
Assembling Construct 2
- 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
- Transform into HeLa cells and see if eGFP is being translated
-> Check if CMV-promoter is working and construct is being read-off completely
- Western Blot
-> Check by reference of protein size if eGFP is cut off from the residual of the protein by TEV-Protease
Contents
Aim 1: DNA replication+PCR
PCR1: replication of the Tet-inducible CMV promotor [X]
PCR2a: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]
PCR2b: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]
PCR3: joining PCR of the TEVrecogn-NDegron-SF3 Part [ ]
PCR4a: replication human bak with mutagenisis (Pst1) [ ]
PCR4b: replication human bak with mutagenisis (Pst1) [ ]
PCR5: joining PCR of human bak [ ]
PCR6: replication of the SV40-polyadenylation site [X]
PCR7a: replication of the p14*TEVrecogn with mutagenisis (Spe1) [ ]
PCR7b: replication of the p14*TEVrecogn with mutagenisis (Spe1) with TEV recogn in primer (16TrecR) [X]
PCR8: joining PCR of p14*TEVrecogn with TEV recogn in primer (16TrecR) [ ]
Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequenz
Aim 3: Assembling Biobricks
Aim 4: Testing products
Construct 1
Construct 2
Aim 5: Testing system