Team:Osaka/Notebook

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==Notebook==
==Notebook==
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===September 12 (Sun)===
 
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# Miniprep of Fcex, 006, 007, 010, 011
 
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# Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
 
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# Gel electrophoresis of digests
 
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#* (RESULTS)
 
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# Ligation for 3A assembly
 
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#* 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
 
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# Transformation of 014 using 2μl ligation product with 50μl competent cells
 
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# Moved yesterday's transformation plates (012, 013) to 4°C refrigerator ''no RFP expression from vector plasmids at all?''
 
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# Transfer of A01, A02, A03 (previously transformed) to solution culture
 
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===September 13 (Mon)===
 
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# Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
 
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#* (RESULTS?)
 
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# PCR test
 
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#* re-cloning of beta-glucosidase from F1 (received from Edinburgh team) into Silver standard-compatible format
 
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#* cloning of pgsC from A01
 
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#* ''note: primers were misdesigned but PCR performed anyway to confirm whether sequences were complementary, and to identify PCR parameters''
 
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# Gel electrophoresis of PCR products
 
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#* (RESULTS?)
 
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# Gel purification of PCR product from F1
 
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# Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
 
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# Gel electrophoresis of PCR product from A01
 
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#* band visible around 400~500bp, which was correct length -> PCR conditions identified!
 
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# Transformation of A01, A02, A03
 
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# Transfer of 012, 013, 014 to solution culture
 
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#* ''RFP expressed from vector plasmids on 012, 013 plates during refrigeration period, so selection of colonies with inserts now possible''
 
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===September 14 (Tue)===
 
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# Miniprep of A02, 012, 013, 014
 
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# Restriction digests
 
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#* 012, 013, 014 with EcoRI, PstI
 
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#* A02 with EcoRI
 
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# Gel electrophoresis of digests
 
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#* A02 was discarded (bad size?)
 
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#* 012~014: digestions repeated with 1μl each of EcoRI, PstI (previous digestions were 0.5μl each)
 
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#* gel electrophoresis of repeat digestions again showed bad sizes for all parts
 
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#** 1-13D terminator part is bad? -> cut check of 1-13D
 
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#** failure to inactivate restriction enzymes before ligations? -> re-ligation
 
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# 1-13D cut check with XbaI, PstI
 
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# Re-ligation (3A assembly)
 
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#* previously digested 009, 008, 011 inactivated at 80°C for 20min before ligation
 
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#* 012: 009 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
 
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#* 013: 008 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
 
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#* 014: 011 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/12)
 
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# Transformation of ligated products (012~014)
 
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# Gel electrophoresis of yesterday's restriction digests of 1-5A and PCR product from F1 followed by their ligation (1-5A as vector, PCR product as insert)
 
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# Transfer of A01, A02, A03 to LB liquid culture medium supplanted with 25μl of 2M glucose to prevent leaky expression from Lac promoter (may be affecting growth)
 
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# Received additional cellulase parts from Karita-sensei: Cel5, Xyn10, Cel44, Man26, Cel8
 
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#* plasmid DNA resuspended in 10μl MiliQ water each
 
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#* transformation with 2μl DNA solution
 
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===September 15 (Wed)===
 
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# Miniprep A02, A03 (''E. coli failed to grow in A01'')
 
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# Cut check of A02, A03 with EcoRI
 
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#* A02 is ok
 
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# Transformation of 3-22G
 
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#* (INFO?)
 
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# PCR cloning of pgsC from A02
 
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# Colony check & transfer of cellulase parts from Karita-sensei to solution culture
 
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# Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]
 
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{|
 
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!Component!!Volume Added!!Final Concentration
 
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|-
 
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|Triton X-100||100μl||2%
 
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|-
 
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|10%SDS||500μl||1%
 
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|-
 
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|5M  NaCl||100μl||100mM
 
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|-
 
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|20mM  Tris-HCl(ph8.0)||2.5ml||10mM
 
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|-
 
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|0.5M  EDTA(ph8.0)||10μl||1mM
 
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|-
 
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|dH2O||1790μl 
 
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|-
 
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|'''TOTAL'''||5ml
 
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|}
 
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# PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase
 
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===September 16 (Thu)===
 
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# Gel electrophoresis to verify yesterday's PCR results
 
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#* pgsA megaprimer: 750bp -> OK
 
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#* pgsB megaprimer: 170bp -> OK
 
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#* pgsC: 450bp -> very faint band?
 
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# Gel extraction of pgsA, pgsB megaprimers
 
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# 2nd step of megaprimer PCR for pgsA, pgsB
 
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#* failure; possible causes:
 
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#** short annealing step?
 
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#** low denaturation temperature?
 
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#** mistakes in procedure?
 
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# PCR
 
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#* pgsC using correct (redesigned) primers
 
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#* pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
 
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#* gel purification of PCR products
 
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# Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
 
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# Cut check of miniprepped parts with XbaI, PstI
 
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# Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
 
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# Gel electrophoresis
 
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#* yeast genome DNA
 
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#* PCR products
 
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#** pgsC -> OK
 
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# Restriction digest
 
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#* pgsC (PCR product) with EcoRI, PstI
 
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#* 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI
 
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===September 17 (Fri)===
 
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# PCR cloning of ADH1 terminator from yeast genome DNA
 
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#* 4 simultaneous attempts with varying template concentration, thermocycle settings & timing of polymerase addition (before or after initial denaturation)
 
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#* gel electrophoresis showed no PCR product obtained for any of the reactions -> ''annealing temperatures were too stringent?''
 
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# PCR of ADH1 terminator (repeat)
 
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#* annealing temperature was lowered from 70°C to 60°C
 
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#* PCR failed again -> ''possible RNA contamination?''
 
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# PCR of ADH1 terminator (2nd repeat)
 
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#* genomic DNA treated with RNase (0.1μl added to 1μl genome DNA; incubation at 37°C for 15min) before using as template
 
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# Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
 
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# Miniprep of 013 and 3-22G
 
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#* 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
 
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#Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
 
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#* (RESULTS?)
 
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# Ligations
 
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#* 018: 3A assembly with 004 as upstream, Fcex as downstream, 1-1C as vector
 
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#* 019: pgsC as insert, 1-1C as vector (''cut at?'')
 
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# PCR cloning of XynA CBM, pgsA
 
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# Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) ''more plasmids needed?''
 
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# Transformation of 004, 005, 008, 009, 018, 019
 
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===September 18 (Sat)===
 
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# PCR cloning of pgsA by overlap extension ''megaprimer method doesn't seem to work so well''
 
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# Gel electrophoresis of PCR product (after overlap step) of pgsA
 
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#* correct band seems to be obtained
 
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# Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
 
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# Miniprep of 006, 007
 
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# Restriction digest with EcoRI, PstI followed by gel electrophoresis
 
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# PCR of pgsB (1st fragment for overlap extension)
 
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#* ''tried 2 times but couldn't get amplification product!''
 
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# Transfer to solution culture: 004, 005, 008, 009, 018, 019
 
===September 19 (Sun)===
===September 19 (Sun)===

Revision as of 09:41, 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 19 (Sun)

  1. PCR of pgsB (repeat)
    • again, no product :(
  2. PCR of pgsB (4th attempt, including yesterday's)
    • no product
  3. Miniprep of 004, 005, 008, 009, 018, 019
  4. Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
  5. 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?
  6. PCR of pgsB 1st fragment (5th attempt)
    • (RESULTS?)
  7. PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
  8. PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
  9. PCR: Phusion activity check using BglX as template & the primers that generated it
  10. PCR: pgsB template check using the outermost primers

September 20 (Mon)

  1. Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
  2. 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?
  3. PCR: primer check
    • pair of primers for each overlap segment were tested
    • (RESULTS?)　
  4. Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
  5. Ligation to make new part 020: pgsA as insert, 1-1C as vector
  6. Transformation of ligation product
  7. 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)

  1. Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
  2. Restriction digest of miniprepped parts with EcoRI, SpeI
  3. 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?
  4. PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
    • band of correct size obtained!
  5. PCR cloning of Man26B, CelB
    • gel run failed to turn up bands; repeat with lower annealing temp
    • repeat run succeeded!
  6. Inoculated YPD liquid culture medium with yeast
  7. New part 020 (contains pgsA) transferred to solution culture
  8. PCR of pgsB (final step - extension of overlapping fragments)
  9. Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone

September 22 (Wed)

  1. Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
  2. Restriction digest of extracted parts with EcoRI, SpeI
  3. Miniprep of 020
  4. Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
  5. Restriction digest of pgsC, pgsA with EcoRI, SpeI
  6. Gel electrophoresis of all digested parts above
    • 020 was bad; repeat ligation?
  7. Transfer of 006, 007 to solution culture (pick up from new colonies?)
  8. 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
  9. Transformation of above ligation products
  10. Extraction of genome DNA from yeast cultured yesterday
  11. PCR cloning of yeast parts from genomic DNA
    • ADH1 terminator
    • ADH2 promoter
    • CYC1 terminator
    • ENO2 promoter
    • SUC2 leader sequence

September 23 (Thu)

  1. Gel electrophoresis of yesterday's PCR products followed by extraction
  2. Restriction digest of PCR products with EcoRI, SpeI
    • ENO2, ADH2 incorrect length -> repeat
  3. PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
  4. Gel electrophoresis of crude PCR product, extraction & purification from gel
    • ENO2 promoter, ADH2 promoter, glr obtained!
  5. PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
  6. Gel electrophoresis of CelB, Man26B, Cel44A PCR products
    • (RESULTS?)
  7. Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
  8. Gel electrophoresis of 006, 007
    • (RESULTS?)
  9. 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)

  1. Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
  2. Extraction from iGEM distribution plates:
IDPart NameResistanceDescription
1-6N<bbpart>BBa_</bbpart>A,KT7 promoter
2-2F<bbpart>BBa_</bbpart>AT7 polymerase
1-6I<bbpart>BBa_</bbpart>Atetracycline-repressible promoter
  1. PCR purification of yesterday's Cel44A
  2. Miniprep of 019, 020
  3. Restriction digest of 019, 020 with XbaI, PstI
    • inserts of correct lengths obtained!
  4. 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 ???
  5. Transformation of ligation products
  6. PCR cloning (repeat) of Man26, CelB
  7. Gel electrophoresis
    • (RESULTS?)

September 25 (Sat)

  1. Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
  2. Restriction digest of miniprepped plasmid DNA with XbaI, PstI (??? these are not biobrick plasmids!) & gel electrophoresis
  3. Transformation of miniprepped parts
  4. Restriction digests
    • 001 with EcoRI, PstI
    • K1 (??) with EcoRI, SpeI
  5. Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
    • 025: xylanase (K1) in 1-1C vector
  6. PCR cloning of CelB
  7. Transformation of 001-2, 025


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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