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From 2010.igem.org

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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