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| | ==Notebook== | | ==Notebook== |
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| - | ===September 26 (Sun)===
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| - | # Transfer to culture solution (yesterday's transformations)
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| - | # Miniprep of 1-6N, 2-2F, 1-6I, 021 ''faint hint of red detected''
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| - | # Restriction digest
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| - | #* 1-6N, 1-6I with EcoRI, SpeI
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| - | #* 2-2F, 021 with XbaI, PstI
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| - | # Gel electrophoresis
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| - | #* (RESULTS?)
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| - | # Miniprep of parts moved to solution culture this morning
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| - | # Cut check with XbaI, PstI <- ''??these are not biobrick plasmids!''
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| - | #* Cel8 - ok
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| - | #* Cel44 - ??
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| - | #* Man26 - ok
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| - | #* Xyn10 - ??
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| - | # Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
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| - | #* 026 - ADH1 terminator
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| - | #* 027 - ADH2 promoter
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| - | #* 028 - CYC1 terminator
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| - | #* 029 - ENO2 promoter
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| - | #* 030 - SUC2 leader sequence
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| - | #* 031 - glr (glutamate racemase)
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| - | # Transformation of ligation products
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| - | # Transfer of yesterday's transformations to solution culture: 001-2, 025
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| - |
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| - | ===September 27 (Mon)===
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| - | # PCR of Man26, CelB
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| - | # PCR of Man48 (??), CBM from XynAcc
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| - | # Restriction digests
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| - | #* 1-2M with EcoRI, SpeI for assembly later
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| - | #* Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (''for checking?'')
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| - | #* 001-2 with EcoRI, SpeI
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| - | #* 025 with XbaI, PstI
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| - | # Gel electrophoresis
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| - | # Ligations
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| - | #* 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
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| - | #* 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
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| - | #* 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (''remake using 001-2'')
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| - | #* 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (<nowiki>" "</nowiki>)
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| - | # Transformation of ligation products
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| - | # Transfer to solution culture: 026, 027, 028, 029, 030, 031
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| - |
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| - | ===September 28 (Tue)===
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| - | # Miniprep of 026, 027, 028, 030, 031
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| - | # Restriction digest
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| - | #* 026, 028, 031 with XbaI, PstI
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| - | #* 027, 030 with EcoRI, SpeI
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| - | # Gel electrophoresis
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| - | #* (RESULTS?)
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| - | # Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
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| - | # Transformation of ligation products as well as 3-2P
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| - | {|
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| - | !ID!!Part Name!!Resistance!!Description
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| - | |-
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| - | |3-2P||<bbpart>BBa_</bbpart>||A||??
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| - | |}
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| - |
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| - | ===September 29 (Wed)===
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| - | # Transfer to solution culture 025, 026, 027, 028, 030, 031
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| - | # Ligations (repeat of 9/26 with different dilution of 1-1C)
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| - | #* 026 - ADH1 terminator
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| - | #* 027 - ADH2 promoter
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| - | #* 028 - CYC1 terminator
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| - | #* 029 - ENO2 promoter
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| - | #* 030 - SUC2 leader sequence
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| - | #* 031 - glr (glutamate racemase)
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| - | # Transformation of 025, 026, 027, 028, 030, 031 (''repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?'')
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| - | # PCR
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| - | #* pgsB - cloning from plasmid
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| - | #* pgsB - amplification from 021
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| - | #* Man28 - amplification from previous product
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| - | # PCR purification of products
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| - | # PCR of CelB, XynA-CBM
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| - | # Miniprep of cultures inoculated this morning (total culture time: 12hr)
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| - | #* 025 -> ok (colorless)
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| - | #* 027 -> turned red; discarded
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| - | #* others: ok?
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| - | # Restriction digest of 026, 028, 031 with XbaI, PstI
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| - | # Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
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| - | # Gel electrophoresis of PCR products
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| - |
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| - | # Restriction digests:
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| - | #* pgsB, Man (??) with EcoRI, SpeI
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| - | #* today's miniprepped plasmids with EcoRI
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| - | #* 1-1C with EcoRI, SpeI
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| - | ===September 30 (Thu)===
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| - | # PCR cloning
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| - | #* CM10
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| - | #* CelB (repeat)
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| - | #* ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
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| - | #* glr (repeat)
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| - | # PCR purification: CM10, XynA-CBM
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| - | # Restriction digest of all above PCR products with EcoRI, SpeI
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| - | # Gel electrophoresis
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| - |
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| - | # Ligation of PCR products to 1-1C backbone
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| - | #* 021: pgsB
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| - | #* 025: XynA-CBM
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| - | #* 026: ADH1 terminator
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| - | #* 028: CYC1 terminator
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| - | #* 031: glr
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| - | #* 034: Man
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| - | #* 035: CM10
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| - | # Transformation of ligation products
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| - |
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| - | # PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
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| - | # Gel electrophoresis
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| - | #* (RESULTS?)
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| - |
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| - | # Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
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| - | # Restriction digest of miniprepped parts
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| - | #* 004, 005, 032 with EcoRI, SpeI (same as earlier today)
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| - | #* 033, 3-2P (same as on 9/29)
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| - | # Gel electrophoresis
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| - | #* (RESULTS?)
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| - |
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| - | ===TO CHECK/CONFIRM===
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| - | * 'K1' (mentioned on 9/250 -> where did it come from?
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| - | * 9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
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| - | * 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)
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| | <html> | | <html> |
| | </div> | | </div> |
| | </html> | | </html> |
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