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__NOTOC__ ==September 19 (Sun)== # PCR of pgsB (repeat) #* again, no product :( # PCR of pgsB (4th attempt, including yesterday's) #* no product # Miniprep of 004, 005, 008, 009, 018, 019 # Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI # Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019 #* apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019 #* (RESULTS?) #* problem with 019? # PCR of pgsB 1st fragment (5th attempt) #* (RESULTS?) # PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension # PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension # PCR: Phusion activity check using BglX as template & the primers that generated it # PCR: pgsB template check using the outermost primers ==September 20 (Mon)== # Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019 # PCR: Phusion polymerase & template checks (repeat of 9/19?) #* positive control (BglX) was amplified -> Phusion polymerase seems to be working ok #* problem with template? primer? thermocycle settings? # PCR: primer check #* pair of primers for each overlap segment were tested #* (RESULTS?)　 # Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI # Ligation to make new part 020: pgsA as insert, 1-1C as vector # Transformation of ligation product # PCR of pgsB 1st fragment (''n-th repeat??'') * '''Note: MANY MANY rounds of PCR carried out today; due to time constraints they are not described here in detail''' ==September 21 (Tue)== # Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red) # Restriction digest of miniprepped parts with EcoRI, SpeI # Gel electrophoresis of digested parts 004, 005, 006, 007, 008, 009, 019 (?) #* 005 - OK #* 014 - no band visible #* 019 - bad length - ''repeat ligation'' #* 006, 007 - bad lengths; ''repeat colony pick-up & culture?'' # PCR to synthesize C-terminal half of pgsB (overlap extension method continued) #* band of correct size obtained! # PCR cloning of Man26B, CelB #* gel run failed to turn up bands; repeat with lower annealing temp #* repeat run succeeded! # Inoculated YPD liquid culture medium with yeast # New part 020 (contains pgsA) transferred to solution culture # PCR of pgsB (final step - extension of overlapping fragments) # Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone ==September 22 (Wed)== # Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification # Restriction digest of extracted parts with EcoRI, SpeI # Miniprep of 020 # Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly) # Restriction digest of pgsC, pgsA with EcoRI, SpeI # Gel electrophoresis of all digested parts above #* ''020 was bad; repeat ligation?'' # Transfer of 006, 007 to solution culture (pick up from new colonies?) # Ligations #* 019: pgsC (PCR product) into 1-1C vector #* 020: pgsA (PCR product) into 1-1C vector #* 021: pgsB (PCR product) into 1-1C vector #* all using PCR products purified today # Transformation of above ligation products # Extraction of genome DNA from yeast cultured yesterday # PCR cloning of yeast parts from genomic DNA #* ADH1 terminator #* ADH2 promoter #* CYC1 terminator #* ENO2 promoter #* SUC2 leader sequence ==September 23 (Thu)== # Gel electrophoresis of yesterday's PCR products followed by extraction # Restriction digest of PCR products with EcoRI, SpeI #* ENO2, ADH2 incorrect length -> repeat # PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase) # Gel electrophoresis of crude PCR product, extraction & purification from gel #* ENO2 promoter, ADH2 promoter, glr obtained! # PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation) # Gel electrophoresis of CelB, Man26B, Cel44A PCR products #* (RESULTS?) # Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI # Gel electrophoresis of 006, 007 #* (RESULTS?) # Transfer to solution culture: 019, 020 #* no white/non-RFP colonies on 021 (pgsB) plate #** insert (PCR product) was not digested properly? #** problem with gel purification? ==September 24 (Fri)== # Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase) # Extraction from iGEM distribution plates: {| !ID!!Part Name!!Resistance!!Description |- |1-6N||<bbpart>BBa_</bbpart>||A,K||T7 promoter |- |2-2F||<bbpart>BBa_</bbpart>||A||T7 polymerase |- |1-6I||<bbpart>BBa_</bbpart>||A||tetracycline-repressible promoter |} # PCR purification of yesterday's Cel44A # Miniprep of 019, 020 # Restriction digest of 019, 020 with XbaI, PstI #* inserts of correct lengths obtained! # Ligations for 3A assembly #* 004: 001 as upstream, 1-2J as downstream, 1-3A as vector #* 005: 001 as upstream, 2-22P as downstream, 1-3A as vector #* pgsB 10xHC 1-3A ''???'' # Transformation of ligation products # PCR cloning (repeat) of Man26, CelB # Gel electrophoresis #* (RESULTS?) ==September 25 (Sat)== # Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei) # Restriction digest of miniprepped plasmid DNA with XbaI, PstI (''??? these are not biobrick plasmids!'') & gel electrophoresis # Transformation of miniprepped parts # Restriction digests #* 001 with EcoRI, PstI #* K1 (''??'') with EcoRI, SpeI # Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2 #* 025: xylanase (K1) in 1-1C vector # PCR cloning of CelB # Transformation of 001-2, 025 [[Team:Osaka/Notebook|Back to Notebook]]

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