SBB11Ntbk-MaryWang

Mary Wang 14:02, 8 March 2011 (EST)

I did an EcoRI/BamHI Digest- I used a master solution which had 5uL of NEB Buffer, , 20uL of ddH2O, 2.5uL of EcoRI, 2.5uL of BamHI then I placed 6uL of the solution in each PCR tubes and placed 4uL of solutions 1-4 in tubes 1-4. Gel 1- lanes 1-4

Used tube 1 in A1 and tube 2 in B2

Mary Wang 13:38, 3 March 2011 (EST)

The plate with ffgfp did not grow. The plate with sbb1139 PGadA showed so a Miniprep was done

Mary Wang 13:38, 1 March 2011 (EST)

Ligation of EcoRI/BamHI

6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 2296 vector, 1uL 1601CA, T4 DNA Ligase

6.5uL ddH2O, 1uL T4 DNA Ligase buffer, 1uL 1139 vector, 1uL 1601KC, T4 DNA Ligase

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)

Today I ran an analytical gel run on part ssb1139:

• Used 2uL product from previous day (PCR ss45f/ss45r on genome MG1655)
• 5uL dye.

This is the 02172011 Analytical Gel2

The analytical gel shows that the part was done incorrectly- I am in lane 5.

I redid the PCR (2x)

• 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

• 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

The PCR was run at 2k45 instead of 2k55

I also digested pBjh1601KA-Bjh2296 using:

• 8uL of eluted PCR product
• 1uL of NEB Buffer 2
• 0.5uL EcoRI
• 0.5uL BamHI.

A prep Gel run- 02172011 Prep Gel2- I am in 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)

Today I did a PCR using oligos ss45r/ss45f on MG1655 genome.

I used Expand polymerase because it was a PCR done on a Genome.

I used the following amounts and put them in the PCR tube in the following order:

• 24uL ddH2O
• 3.3uL 10X expand buffer
• 3.3uL dNTP
• 1uL Oligo 1 (ss45r)
• 1uL Oligo 2 (ss45f)
• 0.5uL Template DNA
• 0.5 Expand polymerase 1

It should be noted that I did not dilute the oligos.

The PCR was run at 2k55.