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

1. ladder
2. 28
3. 29
4. sbb25
5. sbb26
6. sbb32
7. sbb34
8. JG1
9. ZHH 1,2 AG
10. TN 001/002 PT
11. JHA
12. JHB
13. 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 competent cells

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: 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

• 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

1. ladder
2. JG3A
3. JG3B

• notes: 3A could have worked, 3B did not work

Clotho Entry:
Plate A: H2=JG3A,

Team Assay Project

Team 3 Notebook