Team:Osaka/week11
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October 3 (Sun)
- Miniprep of 006, 007, 010, 011, 036, 037, 038 followed by restriction digest with XbaI, PstI
- Restriction digest of yesterday's minipreps: 034, 035, 036 with EcoRI, SpeI
- Gel electrophoresis
- 006 - ok
- 007 - ?
- 010 - bad
- 011 - bad
- 034 - bad
- 035 - ok
- 036 - ok
- 037 - ?
- 038 - not sufficiently digested?
- 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
- 1-2J, 1-18F, 007, 037 with EcoRI, PstI
- Gel electrophoresis
- (RESULTS?)
- Polyacrylamide gel electrophoresis of SUC2 PCR product & elution (recipe/protocol elsewhere)
- elution buffer added & overnight shaking incubation at 37°C
- Cut check of R1, R2 with EcoRI, SpeI
- PCR cloning from yeast genome ADH2, ENO2 again
- Ligation & transformation:
- 039: 001 as upstream, 021 as downstream, 1-5A as vector
- Cut check of PCR products
- results bad; repeat!
- PCR cloning from yeast genome: ADH2 promoter
October 4 (Mon)
- Gel electrophoresis
- sample?
- Transformation og new genes arrived from Utha University.
- HlyA pSB3K3 19.3mg/ml
- ToRA pSB3K3 12.9mg/l
- GeneIII pSB1AK3 197.7mg/ml
- Transfer to soltion culture: 039.
- Restriction digest of 007 with EcoRI.
- Gel electrophoresis: 007.
- Restriction digest of eluted SUC with EcoRI and SpeI.
- 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)
- PCR of glf, Man, ADH2, ENO2, and Gel electrophoresis.
- Transformation of yesterday's ligation products.
- PCR of glf.
- second time?
- Miniprep of 039, 2-4A, 3-11l, R1.
- Restriction digest of R1 with XbaI, PstI, and Man, 039, 2-4A, 3-11l with EcoRI, SpeI.
- Gel electrophoresis of digested plasmids.
- R1 OK
- 039 OK
- 2-4A OK
- 3-11l X
- Man OK
- glr X
- Transfer transformants to solution culture: 1-3A, 1-5A, 1-1C, HlyA, ToRA, GeneIII.
October 6 (Wed)
- Restriction digest of 034(Man) with EcoRI, Spe.
- Transformation of 3-11l, 3-17C, and 2-13N.
- 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)
- Transfer transformants to solution culture: 008, 009, 015, 016, 044, 045, 046.
- Transformation of 3. ligation products.
- More digestion of 034(Man).
- Miniprep of ToRA, HlyA, GeneIII, 1-5A, 1-3A, 1-1C.
- Restriction digest of ToRA, HlyA, GeneIII with XbaI, PstI.
- Gel electrophoresis of 8. samples.
- Restriction digest of 035, 025 with EcoRI, SpeI.
- Gel electrophoresis of 10. samples.
- 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)
- Restriction digest of 1-3A(pSB1C3) with XbaI, PstI to make biobrick constract for sending iGEM front office.
October 7 (Thu)
- PCR
- Transformation of 054(K), 055(K), 056(K), 057(K).
- PCR for Cel44A.
- Miniprep of 008, 009, 015, 016, 044, 045, 046.
- Restriction digest of 015, 016 with EcoRI, SpeI, and 008, 009, 044, 045, 046 with EcoRI, SpeI.
- 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).
- 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
- Transfer transformants to solution culture: 008(K), 009(K), 015(K), 046(K). Re-pick up colony.
- PCR for Cel44A.
- Ligation of 019~025 with pSB1C3 to make biobrick constract.
- 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)
- Restriction digest of PCR products(glr, Cell44) with EcoRI, SpeI.
October 8 (Fri)
- PCR of Cel5B(1st).
- Transformation of Oct. 7 ligation products.
- Because of lacking competent cells, we transformed only parts of samples except 053, 058, 059, 064 and 066.
- Miniprep of 007, 010, 011, 009, 034, 046, 049, 050, 052, 047, 008, 2-13N, 3-11l, 016.
- PCR of Cel5B(2nd).
- Restriction digest of 007, 008, 010, 011, 009, 046, 047, 3-11l, 2-13N, 034, 049, 050, 052, with EcoRI, SpeI.
- 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
- Transformation of 064, 059, 058, 053, 016.
- Restriction digest 008 with EcoRI, SpeI and 016 with EcoRI, PstI.
- Transfar from glycerol stock: 010, 009, 007, 011 to soltuion culture.
- 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.
- Adittional restriction digest of glr, Cel44A, with EcoRI, SpeI.
- Transformation of 1-12M, 1-13D, 1-23L and transfer to soltuion culture.
October 9 (Sat)
- Electrophoresis of Oct. 8 digested products.
- 039 x
- 016 x
- 008 x
- 011 OK
- 022 OK
- 034 Trash
- 3-11I Trash
- 046 OK
- 047 X
- glr X
- Cel44 OK
- PCR of 022.
- Restriction digest of 1-5A, 1-3A, 1-1C with EcoRI, PstI, 026(ADH1 term.) with XbaI, PstI.
- Miniprep of 057, 054 and glycerol stock.
- Transfer to solution culture: 019(C),020(C), 021(C), 022(C), 023(C), 024(C), 025(C), 007(K),013(C).
- Restriction digest of 057, 054 with XbaI, PstI.
- Electrophoresis of Man PCR product and digested product with EcoRI, SpeRI.
- Miniprep of 007, 009, 010, 011.
- Restriction digest of 8. plasmids with EcoRI, SpeI.
- Electrophoresis of 9. samples.
- Transfer transformants to solution culture: 059, 051, 048, 053(Tranfromation in Oct. 6 and Oct. 8), 016, 058.
- Ligation.
- 013: 008(upstream), 1-13D(downstream), 1-5A(vector)
- 056: 035(upstream), 1-13D(downstream), 1-5A(vector)
- 064-2: F3(upstream), 1-13D(downstream), 1-5A(vector)
- 061: 022(upstream), 1-13D(downstream), 1-3A(vector)
- Miniprep of 013, 020-2, 023-2, 024-2.
- Trash: 048, 051, 053, 059.
- Restriction digest of 013, 020-2, 023-2, 024-2 with EcoRI, PstI, 026 with EcoRI, XbaI, 3-2P, 049, 052 with EcoRI, SpeI.
- Electrophoresis of digested products: 3-2D, 052, 049, 026, 024, 023, 020, 013.
- PCR of Cel5B.