SBB10Ntbk-CarolynKwok

Construction Files
Construction of oriO99 Basic Part sbb32

PCR sbb32F and sbb32R on pEC52          	    (536 bp, EcoRI/BamHI) Sub into pBjk2741-Bca1144	           (EcoRI/BamHI, 2170+910, L) Product is pBjk2741-sbb32  		    {oriO99} - sbb32F Forward oligo for cloning of sbb32   ccataGAATTCatgAGATCTagcgcctcagcgcgccgtag sbb32R Reverse oligo for cloning of sbb32   ctgatGGATCCaccaaacgccaacagctcaa

Construction of O99 5'UTR Basic Part sbb24

PCR sbb24F and sbb24R on pEC52          	    (468 bp, EcoRI/BamHI) Sub into pBca9523-Bca1144	           (EcoRI/BamHI, 2472+910, L) Product is pBca9523-sbb24 		    {O99 5'UTR} - sbb24F Forward oligo for cloning of sbb24  ccataGAATTCatgAGATCTacgattctgacgcattttttatg sbb24R Reverse oligo for cloning of sbb24  ctgatGGATCCacgtcaaaaaccagccagaactgcg

2/17/10
Cloning of sbb32 and sbb24 by PCR 1. Oligos sbb32F, sbb32R, sbb24F, sbb24R were diluted to 10uM 2. Reactions were then set up for sbb32 and sbb24 in PCR tubes with the following components: 24 uL ddH20 3.3 uL 10x Expand Buffer 1 uL Oligo 1 1 uL Oligo 2 0.5 uL Expand Polymerase (added last) 0.5 uL Template DNA

3. PCR was run for both parts using the 2K55 program

2/22/10
PCR of sbb24 was repeated because the original PCR product was missing.

An analytical gel was run for sbb32 by Dorothy Tulanont and the results are as follows: lane 1: ladder lane 6: sbb32

An analytical gel was run for sbb24 by Chris Anderson and the results are as follows: lane 1: sbb24 lane 2: ladder

Both parts (sbb24 and sbb32) are at their desired lengths.

Regular Zymo Cleanup of sbb32 1. 180 uL ADB buffer was added to the PCR product, spun through, and discarded 2. PCR product was washed twice with 200 uL of PE buffer 3. PCR product was spun to dry, and eluted with ~33uL ddH20 4. Eluted PCR product was put into Box A

Regular Zymo Cleanup of sbb24 Zymo cleanup was performed by Chris Anderson and the eluted PCR product was put into Box B

2/24/10
EcoRI/BamHI Digest of PCR Products 1. The following reactions were set up for sbb24 and sbb32:

8uL eluted PCR product 1uL NEB Buffer 0.5uL EcoRI 0.5uL BamHI

2. The reactions were mixed and incubated at 37 degrees in the thermocycler for 1 hr 3. Added 10uL digestion to 1uL 6x loading buffer to be loaded into a preparative gel A preparative gel was run for sbb32 and sbb24 by Andrew S and the results are as follows: lane 1: ladder lane 2: sbb24 lane 5: sbb32

Both parts ran off of the gel.

2/26/10
Redo of EcoRI/BamHI digest The digest and preparative gel were completed as described on 2/24/10 A preparative gel was run for sbb32 and sbb24 and the results are as follows: lane 1: ladder lane 2: sbb24 lane 3: sbb32

Both sbb32 and sbb24 are approximately the right length. The extra eluted PCR products were put into box A.

3/1/10
Zymo Gel Purification of sbb24 and sbb32 The parts were purified according to the following procedure: Template:SBB-Protocols Zymo3

Ligation of sbb24 and sbb32 The parts were ligated according to the following procedure: Template:SBB-Protocols Enz4

Transformation of sbb24 and sbb32 Water and KCM were added to the thawed cells. The ligation mixtures were then mixed with the appropriate cells, heat shocked, mixed with 2YT, and then incubated for 1 hr. The cells were then plated on Spec and incubated overnight.

pBca9523-sbb24 was transformed with DH10B cells pBJK2741-sbb32 was transformed with JTK086 cells

3/3/10
Miniprep of sbb24 and sbb32

4 colonies were picked from the plates of both sbb24 and sbb32. The 8 samples of DNA were then purified using the QIAGEN QIAPrep Spin Miniprep kit. The columns containing the miniprepped DNA were labeled sbbew #1-4 and sbb24 #1-4.

3/8/10
Eco/Bam transfer of sbb41 (RFP) 1256 Bjh2247 was used as the source for sbb41 The miniprepped plasmid (1256 Bjh2247) was digested with EcoRI and BamHI. The digest was incubated at 37C for 1 hr and then 80C for 20 min. 0.5 uL T4 DNA ligase was then added to 0.5uL predigest vector and the mixture was incubated at room temp for 30 min. Afterwards sbb41 was transformed into JTK086 cells, rescued and plated.

Mapping of sbb24 and sbb32 lane 1: ladder lane 2: sbb24 #1 lane 3: sbb24 #2 lane 4: sbb24 #3 lane 5: sbb24 #4 lane 6: sbb32 #1 lane 7: sbb32 #2 lane 8: sbb32 #3 lane 9: sbb32 #4

Put sbb24 and sbb32 into clotho and sent them to be sequenced.

3/10/10
Miniprep of sbb41 The part was miniprepped according to the following protocol: Template:SBB-Protocols Micro3

Mapping of sbb41
 * 1) ladder
 * 2) sbb41 A
 * 3) sbb41 B
 * 4) sbb41 C
 * 5) sbb41 D
 * 6) sbb34 A
 * 7) sbb34 B
 * 8) sbb34 C
 * 9) sbb34 D
 * 10) ladder

sbb41 was at the desired length. 2 samples were picked and sent for sequencing.

3/15/10
Sequencing of sbb24 and sbb32 The sequencing for sbb 32 showed good results but the sequencing of sbb24 showed that the clone had 2 point mutations (1 of which was silent). This could have been due to a problem with the template however the 2nd clone was sent for sequencing.

3/17/10
The sequencing for sbb24 clone #2 still had 2 point mutations, however they were at different spots than the first clone. Thus there may have been something wrong with the parent vector. The part will be fixed.

3/29/10
The sequencing for sbb41 was not verified by me but I was informed that the clone was fine.