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From 2010.igem.org

September 12 (Sun)

1. Miniprep of Fcex, 006, 007, 010, 011
2. Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
3. Gel electrophoresis of digests
   • (RESULTS)
4. Ligation for 3A assembly
   • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
5. Transformation of 014 using 2μl ligation product with 50μl competent cells
6. Moved yesterday's transformation plates (012, 013) to 4°C refrigerator no RFP expression from vector plasmids at all?
7. Transfer of A01, A02, A03 (previously transformed) to solution culture

September 13 (Mon)

1. Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
   • (RESULTS?)
2. PCR test
   • re-cloning of beta-glucosidase from F1 (received from Edinburgh team) into Silver standard-compatible format
   • cloning of pgsC from A01
   • note: primers were misdesigned but PCR performed anyway to confirm whether sequences were complementary, and to identify PCR parameters
3. Gel electrophoresis of PCR products
   • (RESULTS?)
4. Gel purification of PCR product from F1
5. Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
6. Gel electrophoresis of PCR product from A01
   • band visible around 400~500bp, which was correct length -> PCR conditions identified!
7. Transformation of A01, A02, A03
8. Transfer of 012, 013, 014 to solution culture
   • RFP expressed from vector plasmids on 012, 013 plates during refrigeration period, so selection of colonies with inserts now possible

September 14 (Tue)

1. Miniprep of A02, 012, 013, 014
2. Restriction digests
   • 012, 013, 014 with EcoRI, PstI
   • A02 with EcoRI
3. Gel electrophoresis of digests
   • A02 was discarded (bad size?)
   • 012~014: digestions repeated with 1μl each of EcoRI, PstI (previous digestions were 0.5μl each)
   • gel electrophoresis of repeat digestions again showed bad sizes for all parts
     • 1-13D terminator part is bad? -> cut check of 1-13D
     • failure to inactivate restriction enzymes before ligations? -> re-ligation
4. 1-13D cut check with XbaI, PstI
5. Re-ligation (3A assembly)
   • previously digested 009, 008, 011 inactivated at 80°C for 20min before ligation
   • 012: 009 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 013: 008 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
   • 014: 011 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/12)
6. Transformation of ligated products (012~014)
7. Gel electrophoresis of yesterday's restriction digests of 1-5A and PCR product from F1 followed by their ligation (1-5A as vector, PCR product as insert)
8. Transfer of A01, A02, A03 to LB liquid culture medium supplanted with 25μl of 2M glucose to prevent leaky expression from Lac promoter (may be affecting growth)
9. Received additional cellulase parts from Karita-sensei: Cel5, Xyn10, Cel44, Man26, Cel8
   • plasmid DNA resuspended in 10μl MiliQ water each
   • transformation with 2μl DNA solution

September 15 (Wed)

1. Miniprep A02, A03 (E. coli failed to grow in A01)
2. Cut check of A02, A03 with EcoRI
   • A02 is ok
3. Transformation of 3-22G
   • (INFO?)
4. PCR cloning of pgsC from A02
5. Colony check & transfer of cellulase parts from Karita-sensei to solution culture
6. Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]

Component	Volume Added	Final Concentration
Triton X-100	100μl	2%
10%SDS	500μl	1%
5M NaCl	100μl	100mM
20mM Tris-HCl(ph8.0)	2.5ml	10mM
0.5M EDTA(ph8.0)	10μl	1mM
dH2O	1790μl
TOTAL	5ml

1. PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase

September 16 (Thu)

1. Gel electrophoresis to verify yesterday's PCR results
   • pgsA megaprimer: 750bp -> OK
   • pgsB megaprimer: 170bp -> OK
   • pgsC: 450bp -> very faint band?
2. Gel extraction of pgsA, pgsB megaprimers
3. 2nd step of megaprimer PCR for pgsA, pgsB
   • failure; possible causes:
     • short annealing step?
     • low denaturation temperature?
     • mistakes in procedure?
4. PCR
   • pgsC using correct (redesigned) primers
   • pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
   • gel purification of PCR products
5. Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
6. Cut check of miniprepped parts with XbaI, PstI
7. Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
8. Gel electrophoresis
   • yeast genome DNA
   • PCR products
     • pgsC -> OK
9. Restriction digest
   • pgsC (PCR product) with EcoRI, PstI
   • 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI

September 17 (Fri)

1. PCR cloning of ADH1 terminator from yeast genome DNA
   • 4 simultaneous attempts with varying template concentration, thermocycle settings & timing of polymerase addition (before or after initial denaturation)
   • gel electrophoresis showed no PCR product obtained for any of the reactions -> annealing temperatures were too stringent?
2. PCR of ADH1 terminator (repeat)
   • annealing temperature was lowered from 70°C to 60°C
   • PCR failed again -> possible RNA contamination?
3. PCR of ADH1 terminator (2nd repeat)
   • genomic DNA treated with RNase (0.1μl added to 1μl genome DNA; incubation at 37°C for 15min) before using as template
4. Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
5. Miniprep of 013 and 3-22G
   • 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
6. Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
   • (RESULTS?)
7. Ligations
   • 018: 3A assembly with 004 as upstream, Fcex as downstream, 1-1C as vector
   • 019: pgsC as insert, 1-1C as vector (cut at?)
8. PCR cloning of XynA CBM, pgsA
9. Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) more plasmids needed?
10. Transformation of 004, 005, 008, 009, 018, 019

September 18 (Sat)

1. PCR cloning of pgsA by overlap extension megaprimer method doesn't seem to work so well
2. Gel electrophoresis of PCR product (after overlap step) of pgsA
   • correct band seems to be obtained
3. Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
4. Miniprep of 006, 007
5. Restriction digest with EcoRI, PstI followed by gel electrophoresis
6. PCR of pgsB (1st fragment for overlap extension)
   • tried 2 times but couldn't get amplification product!
7. Transfer to solution culture: 004, 005, 008, 009, 018, 019

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