SBB10Ntbk-JoseGutierrez

My Project

=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:
 * 1) convert Oligos to 100uM [X(nmol) + 10*X(ul)]
 * 2) dilute stock Oligos to 10uM
 * 3) PCR Procedure

2/17/10
Objective: Run Analytical gel on SBB30 (JG01), after confirmation run zymo cleanup Expected Length (1235 bp) Results:

Dorothy
Lanes PCR product confirmed, continue to Zymo Cleanup
 * 1) ladder
 * 28
 * 29
 * 1) sbb25
 * 2) sbb26
 * 3) sbb32
 * 4) sbb34
 * JG1
 * 1) ZHH 1,2 AG
 * 2) TN 001/002 PT
 * JHA
 * JHB
 * JHC

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 competent cells

3/3/10
pick colonies (already done) Mini Prep picked colonies (4 SBB30, 4 SBB38, 2 SBB37) from plates
 * 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

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: SBB30(1235 bp); SBB 38 (1253, 2170 bp); SBB37(2170, 50 bp) Results:

Jose Gutierrez

 * 1) JG1a(SBB30)
 * 2) JG1b
 * 3) JG1c
 * 4) JG1d
 * 5) JG2a(SBB 38)
 * 6) JG2b
 * 7) Ladder
 * 8) JG2d
 * 9) JG3a(SBB 37)
 * 10) JG3b

Clotho Entry: Plate A: A2=JG1A, H1=JG1B Plate B: A2=JG1B, H1=JG1C
 * notes: SBB37 was too small of a fragment so should have been digested with Eco/BglI not Eco/Bam.

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

 * 1) ladder
 * 2) JG3A
 * 3) JG3B

Clotho Entry: Plate A: H2=JG3A, =Team Assay Project= Team 3 Notebook
 * notes: 3A could have worked, 3B did not work