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Team:Hong Kong CUHK/august
From 2013.igem.org
August
Aug 1
Transformation: M2, M3, QsrR, 23100
Pick clones: Dioxgenase, Laccase from yesterday plates.
Miniprep of Laccase and dioxgenase and gel electrophoresis (no result).
PCR: PolyProline linker; run gel, seemed to be successful, but ladder didn’t look right since bands were missing.
Aug 2
1. Miniprep of Laccase and Dioxgenase
2. PCR of GGGGS linkers and Polyproline linkers
Aug 3
1. Restriction digestion of Laccase and Dioxygenase
2. Run gel: Dioxgenase, Laccase, PCR products
Aug 4
1. Picked colonies from M2, M3, 23100 and QsrR.
2. Miniprep.
3. Run gel, no result.
Aug 5
1. PCR of SsDsbA, TM Helix, Voltage sensor peptide.
2. Run gel
3. Gel clean
4. Ligation
5. Transformation
Aug 6
Pick clones from SsDsbA, TM Helix, Voltage sensor peptide
Transformation of M2, M3, QsrR, 23100
Aug 7
T4 ligation of Dioxygenase, laccase, QsrR, T7+RBS+Dioxygenase, laccase, QsrR.
DNA clean
Transformation
Pick M2, M3, QsrR, 23100
Aug 8
Miniprep of M2, M3, QsrR, 23100 -> run gel -> fail.
Colony-PCR: Dioxygenase, laccase, QsrR -> run gel -> fail.
Transformation of Dioxygenase, laccase, QsrR, T7+RBS+Dioxygenase, laccase, QsrR.
Lab Workshop and talk.
Aug 9
Restriction digestion of PSB1C3, PSB1C3-T7-RBS, Laccase, Dioxgenase, QsrR
Purification with gel purification kit
T4 Ligation
Transformation
Aug 10
Colony PCR on T7-RBS- {dioxgenase, QsrR, PP, GG, VS, TM}.
Run gel
Aug 11
Colony PCR on T7-RBS-{PP, GG, Laccase}, TM
Run gel
Pick colonies from all suspected result colonies
Aug 12
Miniprep on T7-RBS-{PP,GG, QsrR, VS, TM}
Result: QsrR with T7-RBS.
è PSB1C3-T7RBS-QsrR attained.
New round of Biobrick:
Digestion, Ligation, Transformation of PSB1C3+T7RBC+Laccase/Dioxgenase/QsrR
PSB1C3+ PP/GG/VS/TM/Lac/Dioxy/QsrR
Aug 13
Colony PCR on PSB1C3-T70RBS- Lac/Dioxy/QsrR and PSB1C3- VS/PP/GGGGS/TM/Lac/Dioxy/QsrR (no result)
Aug 15
Colony PCR on PSB1C3-T7-RBS-Lac/Dioxy; PSB1C3- VS/TM/Lac/Dioxy/QsrR
è PSB1C3-TM/QsrR got results.
Aug 16
Miniprep: TM+QsrR
Run gel+ digestion.
PCR on Dioxy, Lac, SsDsBa, PDZ domain, SSDsBa PDZ Ligand, SSDsBa PDZ domain+ FL3-Lgt-FL3, SSDsBa PDZ Ligand+FL3-Lgt-FL3
Aug 18
PCR for Dioxygenase, Voltage sensor, TM Helix, PP, GGGGS, PDZ domain+Lgt, +Laccase and PDZ Ligand+ Lgt
Purification, cut, purification, ligation, transformation to PSB1C3 or PSB1C3-T7-RBS
No result
Aug 20
PCR for Dioxygenase, PDZ Domain (With and without Lgt), PDZ Ligand
Purification
Cut Dioxygenase, Domain +/- Lgt, Ligand +/- Lgt, Laccase, Voltage Sensor, TM Helix, PP, GGGGS
Gel Purification for Dioxgenase.
Miniprep M2+M3 for purification.
Aug 21
PCR, cut, ligation, transformation of Di-C, Di-7, La-C, La-7, LL-7, LL-C, V-C, T-C, P-C, G-C: no clone.
Aug 22
PCR DL, D, L with KAPA HiFi Pol
Cut, ligation, transformation of Di-C, Di-7, La-C, La-7, LL-7, LL-C, V-C, T-C, P-C
