"

From 2010.igem.org

September 19 (Sun)

1. PCR of pgsB (repeat)
   • again, no product :(
2. PCR of pgsB (4th attempt, including yesterday's)
   • no product
3. Miniprep of 004, 005, 008, 009, 018, 019
4. Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
5. Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019
   • apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019
   • (RESULTS?)
   • problem with 019?
6. PCR of pgsB 1st fragment (5th attempt)
   • (RESULTS?)
7. PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
8. PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
9. PCR: Phusion activity check using BglX as template & the primers that generated it
10. PCR: pgsB template check using the outermost primers

September 20 (Mon)

1. Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
2. PCR: Phusion polymerase & template checks (repeat of 9/19?)
   • positive control (BglX) was amplified -> Phusion polymerase seems to be working ok
   • problem with template? primer? thermocycle settings?
3. PCR: primer check
   • pair of primers for each overlap segment were tested
   • (RESULTS?)　
4. Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
5. Ligation to make new part 020: pgsA as insert, 1-1C as vector
6. Transformation of ligation product
7. PCR of pgsB 1st fragment (n-th repeat??)

• Note: MANY MANY rounds of PCR carried out today; due to time constraints they are not described here in detail

September 21 (Tue)

1. Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
2. Restriction digest of miniprepped parts with EcoRI, SpeI
3. Gel electrophoresis of digested parts 004, 005, 006, 007, 008, 009, 019 (?)
   • 005 - OK
   • 014 - no band visible
   • 019 - bad length - repeat ligation
   • 006, 007 - bad lengths; repeat colony pick-up & culture?
4. PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
   • band of correct size obtained!
5. PCR cloning of Man26B, CelB
   • gel run failed to turn up bands; repeat with lower annealing temp
   • repeat run succeeded!
6. Inoculated YPD liquid culture medium with yeast
7. New part 020 (contains pgsA) transferred to solution culture
8. PCR of pgsB (final step - extension of overlapping fragments)
9. Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone

September 22 (Wed)

1. Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
2. Restriction digest of extracted parts with EcoRI, SpeI
3. Miniprep of 020
4. Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
5. Restriction digest of pgsC, pgsA with EcoRI, SpeI
6. Gel electrophoresis of all digested parts above
   • 020 was bad; repeat ligation?
7. Transfer of 006, 007 to solution culture (pick up from new colonies?)
8. Ligations
   • 019: pgsC (PCR product) into 1-1C vector
   • 020: pgsA (PCR product) into 1-1C vector
   • 021: pgsB (PCR product) into 1-1C vector
   • all using PCR products purified today
9. Transformation of above ligation products
10. Extraction of genome DNA from yeast cultured yesterday
11. PCR cloning of yeast parts from genomic DNA
    • ADH1 terminator
    • ADH2 promoter
    • CYC1 terminator
    • ENO2 promoter
    • SUC2 leader sequence

September 23 (Thu)

1. Gel electrophoresis of yesterday's PCR products followed by extraction
2. Restriction digest of PCR products with EcoRI, SpeI
   • ENO2, ADH2 incorrect length -> repeat
3. PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
4. Gel electrophoresis of crude PCR product, extraction & purification from gel
   • ENO2 promoter, ADH2 promoter, glr obtained!
5. PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
6. Gel electrophoresis of CelB, Man26B, Cel44A PCR products
   • (RESULTS?)
7. Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
8. Gel electrophoresis of 006, 007
   • (RESULTS?)
9. Transfer to solution culture: 019, 020
   • no white/non-RFP colonies on 021 (pgsB) plate
     • insert (PCR product) was not digested properly?
     • problem with gel purification?

September 24 (Fri)

1. Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
2. Extraction from iGEM distribution plates:

ID	Part Name	Resistance	Description
1-6N	<bbpart>BBa_</bbpart>	A,K	T7 promoter
2-2F	<bbpart>BBa_</bbpart>	A	T7 polymerase
1-6I	<bbpart>BBa_</bbpart>	A	tetracycline-repressible promoter

1. PCR purification of yesterday's Cel44A
2. Miniprep of 019, 020
3. Restriction digest of 019, 020 with XbaI, PstI
   • inserts of correct lengths obtained!
4. Ligations for 3A assembly
   • 004: 001 as upstream, 1-2J as downstream, 1-3A as vector
   • 005: 001 as upstream, 2-22P as downstream, 1-3A as vector
   • pgsB 10xHC 1-3A ???
5. Transformation of ligation products
6. PCR cloning (repeat) of Man26, CelB
7. Gel electrophoresis
   • (RESULTS?)

September 25 (Sat)

1. Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
2. Restriction digest of miniprepped plasmid DNA with XbaI, PstI (??? these are not biobrick plasmids!) & gel electrophoresis
3. Transformation of miniprepped parts
4. Restriction digests
   • 001 with EcoRI, PstI
   • K1 (??) with EcoRI, SpeI
5. Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
   • 025: xylanase (K1) in 1-1C vector
6. PCR cloning of CelB
7. Transformation of 001-2, 025

Back to Notebook