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 a PCR tube 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 2K55

2/22/10

PCR of sbb24 was repeated because the original PCR product was missing.

An analytical gel was run for sbb32 by Dorothy Tulanontand 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

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'''