Team:Osaka/Notebook

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

Revision as of 09:46, 13 October 2010

Calendar

July
Week S M T W T F S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
week1 25 26 27 28 29 30 31
August
Week S M T W T F S
week2 1 2 3 4 5 6 7
week3 8 9 10 11 12 13 14
week4 15 16 17 18 19 20 21
week5 22 23 24 25 26 27 28
week6 29 30 31
September
Week S M T W T F S
week6 1 2 3 4
week7 5 6 7 8 9 10 11
week8 12 13 14 15 16 17 18
week9 19 20 21 22 23 24 25
week10 26 27 28 29 30
October
S M T W T F S
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
November
S M T W T F S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30










Notebook

September 26 (Sun)

  1. Transfer to culture solution (yesterday's transformations)
  2. Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
  3. Restriction digest
    • 1-6N, 1-6I with EcoRI, SpeI
    • 2-2F, 021 with XbaI, PstI
  4. Gel electrophoresis
    • (RESULTS?)
  5. Miniprep of parts moved to solution culture this morning
  6. Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
    • Cel8 - ok
    • Cel44 - ??
    • Man26 - ok
    • Xyn10 - ??
  7. Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
    • 026 - ADH1 terminator
    • 027 - ADH2 promoter
    • 028 - CYC1 terminator
    • 029 - ENO2 promoter
    • 030 - SUC2 leader sequence
    • 031 - glr (glutamate racemase)
  8. Transformation of ligation products
  9. Transfer of yesterday's transformations to solution culture: 001-2, 025

September 27 (Mon)

  1. PCR of Man26, CelB
  2. PCR of Man48 (??), CBM from XynAcc
  3. Restriction digests
    • 1-2M with EcoRI, SpeI for assembly later
    • Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (for checking?)
    • 001-2 with EcoRI, SpeI
    • 025 with XbaI, PstI
  4. Gel electrophoresis
  5. Ligations
    • 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
    • 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
    • 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (remake using 001-2)
    • 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (" ")
  6. Transformation of ligation products
  7. Transfer to solution culture: 026, 027, 028, 029, 030, 031

September 28 (Tue)

  1. Miniprep of 026, 027, 028, 030, 031
  2. Restriction digest
    • 026, 028, 031 with XbaI, PstI
    • 027, 030 with EcoRI, SpeI
  3. Gel electrophoresis
    • (RESULTS?)
  4. Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
  5. Transformation of ligation products as well as 3-2P
IDPart NameResistanceDescription
3-2P<bbpart>BBa_</bbpart>A??

September 29 (Wed)

  1. Transfer to solution culture 025, 026, 027, 028, 030, 031
  2. Ligations (repeat of 9/26 with different dilution of 1-1C)
    • 026 - ADH1 terminator
    • 027 - ADH2 promoter
    • 028 - CYC1 terminator
    • 029 - ENO2 promoter
    • 030 - SUC2 leader sequence
    • 031 - glr (glutamate racemase)
  3. 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?)
  1. PCR
    • pgsB - cloning from plasmid
    • pgsB - amplification from 021
    • Man28 - amplification from previous product
  2. PCR purification of products
  1. PCR of CelB, XynA-CBM
  1. Miniprep of cultures inoculated this morning (total culture time: 12hr)
    • 025 -> ok (colorless)
    • 027 -> turned red; discarded
    • others: ok?
  2. Restriction digest of 026, 028, 031 with XbaI, PstI
  3. Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
  4. Gel electrophoresis of PCR products
  1. Restriction digests:
    • pgsB, Man (??) with EcoRI, SpeI
    • today's miniprepped plasmids with EcoRI
    • 1-1C with EcoRI, SpeI

September 30 (Thu)

  1. PCR cloning
    • CM10
    • CelB (repeat)
    • ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
    • glr (repeat)
  2. PCR purification: CM10, XynA-CBM
  3. Restriction digest of all above PCR products with EcoRI, SpeI
  4. Gel electrophoresis
  1. Ligation of PCR products to 1-1C backbone
    • 021: pgsB
    • 025: XynA-CBM
    • 026: ADH1 terminator
    • 028: CYC1 terminator
    • 031: glr
    • 034: Man
    • 035: CM10
  2. Transformation of ligation products
  1. PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
  2. Gel electrophoresis
    • (RESULTS?)
  1. Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
  2. Restriction digest of miniprepped parts
    • 004, 005, 032 with EcoRI, SpeI (same as earlier today)
    • 033, 3-2P (same as on 9/29)
  3. Gel electrophoresis
    • (RESULTS?)

TO CHECK/CONFIRM

  • 'K1' (mentioned on 9/250 -> where did it come from?
  • 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?
  • 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)

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