SBB11Ntbk-MaryWang

Mary Wang 13:20, 24 February 2011 (EST)

EcoRI/BamHI Digest on pBjh1601KA-Bjh2296

Run Prep Gel

Zymo Gel Clean Up

elute with 8uL ddH2O

Mary Wang 13:20, 23 February 2011 (EST)

EcoRI, BamHI digest on PCR ss45f/ss45r on MG1655 gen. Prep gel on PCR. Showed up on analytical gel 4- lane 1

Mary Wang 22:47, 18 February 2011 (EST)

Gel zymo purification done on the digest of pBjh1601KA- labeled MW8

2X 200uL of PE buffer. Eluted with 8uL of ddH2O.

Analytical gel run on PCR on part sbb1139

Analytical Gel1 Lane 3 and 4 (Lane 3 is no DMSO, Lane 4 has DMSO)

DMSO PCR did not show up in Lane 4

Regular Zymo cleanup done- labeled MW9

180uL of ADB buffer, 2X 200uL PE buffer- elute with 31uL of ddH2O (2uL used for PCR)

Mary Wang 14:18, 17 February 2011 (EST)

Analytical gel run on part ssb1139: Used 2uL product from previous day and 5uL dye. Analytical gel shows that the part was done incorrectly. Analytical Gel2- lane 5.

REdone (x2)

One: 24uL ddH2O, 3.3uL 10X expand buffer, 3.3 uL dNTP, 1 uL ss45r, 1uL ss45f, 0.5uL template DNA, 0.5 expand polymerase 1

Two: 21.7uL ddH2O, 3.3uL DMSO, 3.3uL 10X expand buffer, 3.3uL dNTP, 1uLss45r, 1uL ss45f, 0.5uL template DNA, 0.5 expand polymerase 1

Run at 2k45 instead of 2k55

Digest pBjh1601KA-Bjh2296 using: 8uL of eluted PCR product, 1uL of NEB Buffer 2, 0.5uL EcoRI and 0.5uL BamHI. Prep Gel run- shown on PrepGel2- lane 4. Cut out ~837 part- melted with 600uL ADB buffer, melt at 55C for 10 minutes then frozen.

Mary Wang 14:16, 15 February 2011 (EST)

PCR ss45r/ss45f on MG1655 genome.

Used Expand polymerase because it was a genome.

Put template DNA into PCR tube first then the Expand polymerase second.

Did not dilute the oligos.

Used cloning by PCR- basic PCR method.

Amounts used: 24uL ddH2O, 3.3uL 10X expand buffer, 3.3uL dNTP, 1uL Oligo 1, 1uL Oligo 2, 0.5 Expand polymerase 1, 0.5uL Template DNA

Run at 2k55