Team:Osaka/week7

From 2010.igem.org

September 5 (Sun)

  1. Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.

September 6 (Mon)

  1. Transfer of yesterday's transformations to solution culture
  2. Transformation of the following registry parts (See Table 8)
Table 8
IDPart NameResistanceDescription
2-10FBBa_K081005Aconstitutive promoter from combinatorial library + RBS
2-10HBBa_K081006Alambda phage promoter + RBS

September 7 (Tue)

  1. Miniprep of 004, 005
  2. Cut check with EcoRI, SpeI
    • both insert lengths ok!
  3. Transformation of DNA for PGA synthesis-related genes (See Table 9)
  4. Transfer to solution culture
    • 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
    • 2-10F, 2-10H transformed yesterday
Table 9
IDPart NameResistanceDescription
A01pTPG01-1Aplasmid pTrc99A with pgs genes inserted
A02pTPG01-2A''
A03pBSGR3Kglutamine racemase

September 8 (Wed)

  1. Miniprep 2-10F, 2-10H, 004, 005
  2. Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
  3. Gel electrophoresis
    • new batch of EtBr for staining
    • (RESULTS?)
  4. Transfer of A01~A03 to solution culture
  5. PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara Ex Taq polymerase kit
    • it took several tries to get a successful reaction
      • 1st attempt: template DNA was used directly; concentration too high (failure)?
      • 2nd attempt: 100X, 1000X dilutions attempted without success; this time, over-dilution or stringent annealing temp (68°C) may have been culprit?
      • 3rd attempt: 10X dilutions, annealing temp lowered to 65°C -> success!
    • note: reactions were evaluated by gel electrophoresis of crude PCR product - if band appears at approximately correct length then reaction judged as successful
    • special note of thanks to Nakamura who stayed in lab overnight to run the PCRs

September 9 (Thu)

  1. Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
  2. Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
  3. Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
  4. Gel electrophoresis of digested parts together with 1-5A 1-5A supposed to be receiving vector, but digested at wrong sites
    • CenA PCR product -> OK (Silver-compatible part designated FcenA)
    • Cex PCR product -> ?
  5. Another round of PCR to amplify Cex as Silver standard part (why?)
    • 10X dilution of template
    • 68°C annealing temp
  6. Ligation
    • FcenA: PCR product with 1-5A as vector (cut/ligated at X, P)
    • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
    • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
    • 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) bad insert?
    • 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
  7. Transformation of newly assembled parts 006~009
  8. Transfer of 006~009 to solution culture.

September 10 (Fri)

  1. Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
    • PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
  2. Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
    • (RESULTS?)
  3. Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with XbaI, PstI followed by gel electrophoresis
    • (RESULTS?)
  4. Ligations
    • Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
    • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector); repeat
    • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); repeat
    • 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)
    • 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
  5. Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
  6. Colony check of 9/9 transformations
    • 006: no colonies
    • 007: no colonies
    • 008: >100 colonies bad insert?
    • 009: >100 colonies
    • FcenA: >100 colonies
  7. Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (more 001, 2-10F needed)

September 11 (Sat)

  1. Miniprep of 008, 009, FcenA, 001, 2-10F.
  2. Ristriction digest of 008, 009, 001, 2-10F with EcoRI, SpeI and FcenA with XbaI, PstI.
  3. Gel electrophoresis of digested 008, 009, FcenA, 001, 2-10F.
    • 008 -> ???
    • 009 -> O.K.
    • add 1-13D as terminator to 008 and 009'
    • FcenA was not digested by XbaI
  4. Restriction digest of FcenA with EcoRI.
  5. Gel electrophoresis of FcenA
    • FcenA was digested by EcoRI -> O.K.
  6. Ligations for 3A assembly
    • 012: 009 (upstream), 1-13D(terminator, downstream), 1-3A (vector)
    • 013: 008 (upstream), 1-13D(terminator, downstream), 1-5A (vector) bad insert?
  7. Transfomation of newly assembled parts 012, 013
  8. Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.


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