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.

October 6 (Wed)

  1. Restriction digest of 034(Man) with EcoRI, Spe.
  2. Transformation of 3-11l, 3-17C, and 2-13N.
  3. Ligation
    • 047: 039(upstream), 036(downstream), 1-1C(vector)
    • 010: 004(upstream), 023(downstream), 1-1C(vector)
    • 011: 005(upstream), Fcex 023(downstream), 1-1C(vector)
    • 034: Man, 1-3A(vector)
    • 048: R1(upstream), FcenA 022(downstream), 1-3A(vector)
    • 049: R1(upstream), Fcex 023(downstream), 1-3A(vector)
    • 051: R2(upstream), 022(downstream), 1-3A(vector)
    • 052: R2(upstream), 023(downstream), 1-3A(vector)
    • 053: R2(upstream), 024(downstream), 1-3A(vector)
  4. Transfer transformants to solution culture: 008, 009, 015, 016, 044, 045, 046.
  5. Transformation of 3. ligation products.
  6. More digestion of 034(Man).
  7. Miniprep of ToRA, HlyA, GeneIII, 1-5A, 1-3A, 1-1C.
  8. Restriction digest of ToRA, HlyA, GeneIII with XbaI, PstI.
  9. Gel electrophoresis of 8. samples.
  10. Restriction digest of 035, 025 with EcoRI, SpeI.
  11. Gel electrophoresis of 10. samples.
  12. Ligation
    • 054(K): 035(upstream), 026(downstream), 1-5A(vector)
    • 055(K): 025(upstream), 026(downstream), 1-5A(vector)
    • 056(K): 035(upstream), 1-13D(downstream), 1-5A(vector)
    • 057(K): 025(upstream), 1-13D(downstream), 1-5A(vector)
  13. Restriction digest of 1-3A(pSB1C3) with XbaI, PstI to make biobrick constract for sending iGEM front office.

October 7 (Thu)

  1. PCR
  2. Transformation of 054(K), 055(K), 056(K), 057(K).
  3. PCR for Cel44A.
  4. Miniprep of 008, 009, 015, 016, 044, 045, 046.
  5. Restriction digest of 015, 016 with EcoRI, SpeI, and 008, 009, 044, 045, 046 with EcoRI, SpeI.
  6. Transfer transformants to solution culture: 047, 011, 010, 034(C), 049(C), 050(C), 052(C), 3-11l, 2-13N.
    • Because of failing, re-Ligation of 047(C), 048(C), 051(C), 053(C).
  7. Gel electrophoresis of 5. samples.
    • 008 OK
    • 009 X
    • 015 X
    • 016 compare with only EcoRI digest
    • 044 compare with only EcoRI digest
    • 045 OK
    • 046 X
    • 1-3A OK
  8. Transfer transformants to solution culture: 008(K), 009(K), 015(K), 046(K). Re-pick up colony.
  9. PCR for Cel44A.
  10. Ligation of 019~025 with pSB1C3 to make biobrick constract.
  11. Ligation
    • 058: 001(upstream), HlyA(downstream), 1-3A(vector)
    • 059: 001(upstream), ToRA(downstream), 1-3A(vector)
    • 060: 001(upstream), GeneIII(downstream), 1-3A(vector)
    • 061: 022(upstream), 1-13D(downstream), 1-3A(vector)
    • 062: 023(upstream), 1-13D(downstream), 1-3A(vector)
    • 063: 024(upstream), 1-13D(downstream), 1-3A(vector)
    • 064: 044(upstream), 1-13D(downstream), 1-5A(vector)
    • 065: 045(upstream), 1-13A(downstream), 1-3A(vector)
    • 009: 001(upstream), 2-20J(downstream), 1-5A(vector)
    • 013: 008(upstream), 1-13D(downstream), 1-3A(vector)
    • 016: 007(upstream), 1-13D(downstream), 1-3A(vector)
    • 047: 039(upstream), 036(downstream), 1-1C(vector)
    • 048: RI(upstream), 022(downstream), 1-3A(vector)
    • 051: R2(upstream), 022(downstream), 1-3A(vector)
    • 053: R2(upstream), 024(downstream), 1-3A(vector)
  12. Restriction digest of PCR products(glr, Cell44) with EcoRI, SpeI.

October 8 (Fri)

  1. PCR of Cel5B(1st).
  2. Transformation of Oct. 7 ligation products.
    • Because of lacking competent cells, we transformed only parts of samples except 053, 058, 059, 064 and 066.
  3. Miniprep of 007, 010, 011, 009, 034, 046, 049, 050, 052, 047, 008, 2-13N, 3-11l, 016.
  4. PCR of Cel5B(2nd).
  5. Restriction digest of 007, 008, 010, 011, 009, 046, 047, 3-11l, 2-13N, 034, 049, 050, 052, with EcoRI, SpeI.
  6. Gel electrophoresis of digested products.
    • 052 OK
    • 050 OK
    • 049 OK
    • 034 Man is X
    • 011 X More PstI digest.
    • 010 OK
    • 009 OK
    • 008 OK
    • 007 OK
    • 2-13N OK
    • 3-11J  ? Trash
    • 047 X
    • 046 Only PstI digest.
    • 034 X
  7. Transformation of 064, 059, 058, 053, 016.
  8. Restriction digest 008 with EcoRI, SpeI and 016 with EcoRI, PstI.
  9. Transfar from glycerol stock: 010, 009, 007, 011 to soltuion culture.
  10. Restriction digest of 046, 011, 034 with PstI, 047, 3-11l with EcoRI, 022 with EcoRI, SpeI, 036 with XbaI, PstI, and 039 with EcoRI, SpeI.
  11. Adittional restriction digest of glr, Cel44A, with EcoRI, SpeI.
  12. Transformation of 1-12M, 1-13D, 1-23L and transfer to soltuion culture.

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