"

From 2010.igem.org

---

Revision as of 09:51, 13 October 2010

Contents 1 September 26 (Sun) 2 September 27 (Mon) 3 September 28 (Tue) 4 September 29 (Wed) 5 September 30 (Thu)

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

ID	Part Name	Resistance	Description
3-2P	<bbpart>BBa_</bbpart>	A	??

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?)