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From 2010.igem.org

September 5 (Sun)

1. Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.

September 6 (Mon)

1. Transfer of yesterday's transformations to solution culture
2. Transformation of the following registry parts (See Table 8)

Table 8

ID	Part Name	Resistance	Description
2-10F	<bbpart>BBa_K081005</bbpart>	A	constitutive promoter from combinatorial library + RBS
2-10H	<bbpart>BBa_K081006</bbpart>	A	lambda phage promoter + RBS

September 7 (Tue)

1. Miniprep of 004, 005
2. Cut check with EcoRI, SpeI
   • both insert lengths ok!
3. Transformation of DNA for PGA synthesis-related genes (See Table 9)
4. Transfer to solution culture
   • 004, 005 transformed on 9/5 (pick up from fresh colonies) -> needed more plasmid
   • 2-10F, 2-10H transformed yesterday

Table 9

ID	Part Name	Resistance	Description
A01	pTPG01-1	A	plasmid pTrc99A with pgs genes inserted
A02	pTPG01-2	A	''
A03	pBSGR3	K	glutamine racemase

September 8 (Wed)

1. Miniprep 2-10F, 2-10H, 004, 005
2. Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
3. Gel electrophoresis
   • new batch of EtBr for staining
   • (RESULTS?)
4. Transfer of A01~A03 to solution culture
5. PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara Ex Taq polymerase kit
   • it took several tries to get a successful reaction
     • 1st attempt: template DNA was used directly; concentration too high (failure)?
     • 2nd attempt: 100X, 1000X dilutions attempted without success; this time, over-dilution or stringent annealing temp (68°C) may have been culprit?
     • 3rd attempt: 10X dilutions, annealing temp lowered to 65°C -> success!
   • note: reactions were evaluated by gel electrophoresis of crude PCR product - if band appears at approximately correct length then reaction judged as successful
   • special note of thanks to Nakamura who stayed in lab overnight to run the PCRs

September 9 (Thu)

1. Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
2. Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (why not use PCR purification kit??)
3. Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
4. Gel electrophoresis of digested parts together with 1-5A 1-5A supposed to be receiving vector, but digested at wrong sites
   • CenA PCR product -> OK (Silver-compatible part designated FcenA)
   • Cex PCR product -> ?
5. Another round of PCR to amplify Cex as Silver standard part (why?)
   • 10X dilution of template
   • 68°C annealing temp
6. Ligation
   • FcenA: PCR product with 1-5A as vector (cut/ligated at X, P)
   • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
   • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
   • 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) bad insert?
   • 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
7. Transformation of newly assembled parts 006~009
8. Transfer of 006~009 to solution culture.

September 10 (Fri)

1. Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
   • PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
2. Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
   • (RESULTS?)
3. Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with　XbaI, PstI followed by gel electrophoresis
   • (RESULTS?)
4. Ligations
   • Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
   • 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector); repeat
   • 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); repeat
   • 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)
   • 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
5. Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
6. Colony check of 9/9 transformations
   • 006: no colonies
   • 007: no colonies
   • 008: >100 colonies bad insert?
   • 009: >100 colonies
   • FcenA: >100 colonies
7. Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (more 001, 2-10F needed)

September 11 (Sat)

1. Miniprep of 008, 009, FcenA, 001, 2-10F.
2. Ristriction digest of 008, 009, 001, 2-10F with EcoRI,　SpeI and FcenA with XbaI, PstI.
3. Gel electrophoresis of digested 008, 009, FcenA, 001, 2-10F.
   • 008 -> ???
   • 009 -> O.K.
   • add 1-13D as terminator to 008 and 009'
   • FcenA was not digested by XbaI
4. Restriction digest of FcenA with EcoRI.
5. Gel electrophoresis of FcenA
   • FcenA was digested by EcoRI -> O.K.
6. Ligations for 3A assembly
   • 012: 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
   • 013: 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector) bad insert?
7. Transfomation of newly assembled parts 012, 013
8. Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.

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