SBB10Ntbk-JoseGutierrez
Construction Files
SBB30: 5'UTR_rep009
Biobricking of O99 rep Gene PCR JG001-F and JG002-R on pEC52 (1235 bp, EcoRI/BamHI) Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L) Product is pBca9523-sbb30 {5'UTR_repO99!} ---------------------------- JG001-F Cloning of O99 rep Gene ccataGAATTCatgAGATCTaaaaacgattctgacgcattttttatg JG002-R Cloning of O99 rep Gene CTGATGGATCCCTACTCTACAAGACCTCGTTTTttc
SBB38: PBad Promoter
Eco/Bam transfer pBjh1601CK-ig114 Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-ig114 {AraC-Pbad}
SBB37: PCon Promoter
Eco/Bam transfer pBca1100-Bca1152 Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-Bca1152 {Pcon-J23100}
Parts Assembly
2/17/10
Objective: Set up PCR reaction for biobricking of SBB30 009 rep Gene -PCR JG 001-F & JG002-R on pEC52 (1235 bp Eco/Bam)
Procedure:
- convert Oligos to 100uM [X(nmol) + 10*X(ul)]
- dilute stock Oligos to 10uM
- PCR Procedure
2/17/10
Objective: Run Analytical gel on SBB30 (JG01), after confirmation run zymo cleanup
Results:
Dorothy
- ladder
- 28
- 29
- sbb25
- sbb26
- sbb32
- sbb34
- JG1
- ZHH 1,2 AG
- TN 001/002 PT
- JHA
- JHB
- JHC
PCR product confirmed, continue to Zymo Cleanup
2/24/10
Eco/Bam Digest of SBB30 PCR products
3/1/10
To Do List:
Zymo gell purification of SBB30 Eco/Bam Digest
Eco/Bam transfer pBjh1601CK-ig114
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-ig114 {AraC-Pbad}
Eco/Bam transfer pBca1100-Bca1152
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-Bca1152 {Pcon-J23100}
Ligation's:
SBB30 (vector:pBca9523, start: 2:35, end: 3:07)
SBB38 (vector:pBjk2741, start: 3:10, end: 3:40)
SBB37 (vector:pBjk2741, start: 3:10, end: 3:40)
Transformation of Ligation products into ________
3/3/10
pick colonies (already done)
- Note: SBB 37 was mostly red colonies on plate only two possible good colonies on plate, probably due to re-ligation of original vector also SBB37 is small which might make Eco/Bam transfer fail, other plates looked good
Mini Prep picked colonies (4 SBB30, 4 SBB38, 2 SBB37) from plates
3/8/10
To Do: Run analytical gel of miniprep plasmids
- Eco/Bam Digest of miniprep plasmids
-run analytical get of digests, Expected product lengths (XXX bp)
Results:
Jose Gutierrez
- JG1a
- JG1b
- JG1c
- JG1d
- JG2a
- JG2b
- Ladder
- JG2d
- JG3a
- JG3b
- notes: SBB37 was too small of a fragment so should have been digested with Eco/BglI not Eco/Bam.
Clotho Entry:
Plate A: A2=JG1A, H1=JG1B
Plate B: A2=JG1B, H1=JG1C
3/10/10
To Do: Run analytical gel of miniprep plasmids (JG3A,JG3B)
- Eco/BglI Digest of miniprep plasmids <br.
-run analytical get of digests, Expected product length (1536/ 684 bp)
Results:
Jose Gutierrez
- ladder
- JG3A
- JG3B
- notes: 3A could have worked, 3B did not work
Clotho Entry:
Plate A: H2=JG3A,