Team:Osaka/week11

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October 3 (Sun)

  1. Miniprep of 006, 007, 010, 011, 036, 037, 038 followed by restriction digest with XbaI, PstI
  2. Restriction digest of yesterday's minipreps: 034, 035, 036 with EcoRI, SpeI
  3. Gel electrophoresis
    • 006 - ok
    • 007 - ?
    • 010 - bad
    • 011 - bad
    • 034 - bad
    • 035 - ok
    • 036 - ok
    • 037 - ?
    • 038 - not sufficiently digested?
  4. Another round of restriction digest:
    • spare samples of 010, 011, 037 with XbaI, PstI as above (2 colonies were cultured in solution & miniprepped)
    • 038: more XbaI, PstI added to previous tube
  5. 1-2J, 1-18F, 007, 037 with EcoRI, PstI
  6. Gel electrophoresis
    • (RESULTS?)
  7. Polyacrylamide gel electrophoresis of SUC2 PCR product & elution (recipe/protocol elsewhere)
    • elution buffer added & overnight shaking incubation at 37°C
  8. Cut check of R1, R2 with EcoRI, SpeI
  9. PCR cloning from yeast genome ADH2, ENO2 again
  10. Ligation & transformation:
    • 039: 001 as upstream, 021 as downstream, 1-5A as vector
  11. Cut check of PCR products
    • results bad; repeat!
  12. PCR cloning from yeast genome: ADH2 promoter

October 4 (Mon)

  1. Gel electrophoresis
    • sample?
  2. Transformation og new genes arrived from Utha University.
    • HlyA pSB3K3 19.3mg/ml
    • ToRA pSB3K3 12.9mg/l
    • GeneIII pSB1AK3 197.7mg/ml
  3. Transfer to soltion culture: 039.
  4. Restriction digest of 007 with EcoRI.
  5. Gel electrophoresis: 007.
  6. Restriction digest of eluted SUC with EcoRI and SpeI.
  7. Ligation
    • 008(K): 001(upstream), 2-20H(downstream), 1-5A(vector)
    • 009(K): 001(upstream), 2-20J(downstream), 1-5A(vector)
    • 010(K): 004(upstream), Fcex(023, downstream), 1-5A(vector)
    • 015(C): 001(upstream), 1-13D(downstream), 1-3A(vector)
    • 016(C): 007(upstream), 1-13D(downstream), 1-3A(vector)
    • 044(C): 001-2(upstream), F1(downstream), 1-3A(vector)
    • 045(A): 005(upstream), 024(beta-gluctosidase, downstream), 1-1C(vector)
    • 046(A): 004(upstream), 024(downstream), 1-1C(vector)

October 5 (Tue)

  1. PCR of glf, Man, ADH2, ENO2, and Gel electrophoresis.
  2. Transformation of yesterday's ligation products.
  3. PCR of glf.
    • second time?
  4. Miniprep of 039, 2-4A, 3-11l, R1.
  5. Restriction digest of R1 with XbaI, PstI, and Man, 039, 2-4A, 3-11l with EcoRI, SpeI.
  6. Gel electrophoresis of digested plasmids.
    • R1 OK
    • 039 OK
    • 2-4A OK
    • 3-11l X
    • Man OK
    • glr X
  7. Transfer transformants to solution culture: 1-3A, 1-5A, 1-1C, HlyA, ToRA, GeneIII.


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