Team:Osaka/Notebook

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Revision as of 16:59, 14 October 2010

Calendar

Notebook

October 1 (Fri)

1. Gel electrophoresis (?)
2. Ligations
   • 006: 004 as upstream, FcenA as downstream, 1-5A as vector
   • 007: 005 as upstream, FcenA as downstream, 1-5A as vector
   • 010: 004 as upstream, Fcex as downstream, 1-5A as vector
   • 011: 005 as upstream, Fcex as downstream, 1-5A as vector
   • 036: 032 as upstream, 033 as downstream, 1-5A as vector
   • 037: 004 as upstream, 024 as downstream, 1-1C as vector
   • 038: 005 as upstream, 024 as downstream, 1-1C as vector
3. Transformation of ligation products
4. PCR of ENO2 promoter, ADH2 promoter, SUC2 signal sequence
5. Parts from NYU team:

1. Miniprep of 026, 028, 030
2. Restriction digest
   • 026, 028 with XbaI, PstI
   • 030 with EcoRI, SpeI or EcoRI only
3. Gel electrophoresis
4. (RESULTS?)
5. Transfer to solution culture: 021, 025, 026, 034, 035

October 2 (Sat)

1. Restriction digest of plasmid backbones: 1-5A, 1-3A with XbaI, PstI
2. Electrophoresis & purification from gel
3. PCR of SUC2 signal sequence
   • primers diluted with MiliQ water from 100μM to 5μM
   • Taq polymerase added before starting (as opposed to after initial denaturation step)
   • gel run to check -> (RESULTS?)
4. PCR cloning from yeast genome ENO2, ADH2 promoters
5. Transfer to culture solution: 006, 007, 010, 011, 036, 037, 038
6. Miniprep of 021, 025, 026, 034, 035 followed by restriction digest with XbaI, PstI
7. Gel electrophoresis
   • 021 - OK
   • 025 - OK
   • 026 - OK
   • 034 - bad
   • 035 - OK

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)

TO CHECK/CONFIRM

• 'K1' (mentioned on 9/250 -> where did it come from?
• 9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
• 9/24~27 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)