Team:Osaka/week10

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Revision as of 16:45, 16 October 2010

September 26 (Sun)

1. Transfer to culture solution (yesterday's transformations)
2. Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
3. Restriction digest
   • 1-6N, 1-6I with EcoRI, SpeI
   • 2-2F, 021 with XbaI, PstI
4. Gel electrophoresis
   • (RESULTS?)
5. Miniprep of parts moved to solution culture this morning
6. Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
   • Cel8 - ok
   • Cel44 - ??
   • Man26 - ok
   • Xyn10 - ??
7. Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
   • 026 - ADH1 terminator
   • 027 - ADH2 promoter
   • 028 - CYC1 terminator
   • 029 - ENO2 promoter
   • 030 - SUC2 leader sequence
   • 031 - glr (glutamate racemase)
8. Transformation of ligation products
9. Transfer of yesterday's transformations to solution culture: 001-2, 025

September 27 (Mon)

1. PCR of Man26, CelB
2. PCR of Man48 (??), CBM from XynAcc
3. Restriction digests
   • 1-2M with EcoRI, SpeI for assembly later
   • Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (for checking?)
   • 001-2 with EcoRI, SpeI
   • 025 with XbaI, PstI
4. Gel electrophoresis
5. Ligations
   • 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
   • 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
   • 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (remake using 001-2)
   • 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (" ")
6. Transformation of ligation products
7. Transfer to solution culture: 026, 027, 028, 029, 030, 031

September 28 (Tue)

1. Miniprep of 026, 027, 028, 030, 031
2. Restriction digest
   • 026, 028, 031 with XbaI, PstI
   • 027, 030 with EcoRI, SpeI
3. Gel electrophoresis
   • (RESULTS?)
4. Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
5. Transformation of ligation products as well as 3-2P

September 29 (Wed)

1. Transfer to solution culture 025, 026, 027, 028, 030, 031
2. Ligations (repeat of 9/26 with different dilution of 1-1C)
   • 026 - ADH1 terminator
   • 027 - ADH2 promoter
   • 028 - CYC1 terminator
   • 029 - ENO2 promoter
   • 030 - SUC2 leader sequence
   • 031 - glr (glutamate racemase)
3. Transformation of 025, 026, 027, 028, 030, 031 (repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?)

1. PCR
   • pgsB - cloning from plasmid
   • pgsB - amplification from 021
   • Man28 - amplification from previous product
2. PCR purification of products

1. PCR of CelB, XynA-CBM

1. Miniprep of cultures inoculated this morning (total culture time: 12hr)
   • 025 -> ok (colorless)
   • 027 -> turned red; discarded
   • others: ok?
2. Restriction digest of 026, 028, 031 with XbaI, PstI
3. Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
4. Gel electrophoresis of PCR products

1. Restriction digests:
   • pgsB, Man (??) with EcoRI, SpeI
   • today's miniprepped plasmids with EcoRI
   • 1-1C with EcoRI, SpeI

September 30 (Thu)

1. PCR cloning
   • CM10
   • CelB (repeat)
   • ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
   • glr (repeat)
2. PCR purification: CM10, XynA-CBM
3. Restriction digest of all above PCR products with EcoRI, SpeI
4. Gel electrophoresis

1. Ligation of PCR products to 1-1C backbone
   • 021: pgsB
   • 025: XynA-CBM
   • 026: ADH1 terminator
   • 028: CYC1 terminator
   • 031: glr
   • 034: Man
   • 035: CM10
2. Transformation of ligation products

1. PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
2. Gel electrophoresis
   • (RESULTS?)

1. Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
2. Restriction digest of miniprepped parts
   • 004, 005, 032 with EcoRI, SpeI (same as earlier today)
   • 033, 3-2P (same as on 9/29)
3. Gel electrophoresis
   • (RESULTS?)

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)

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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