SBB11Ntbk-JosephSilo

Joseph Silo 18:06, 6 March 2011 (EST)

March 3. Colonies were already picked and innoculated when I arrived to lab. There were four replicates of each part. The Miniprep protocol was used.

Joseph Silo 18:03, 6 March 2011 (EST)

Ligation and transformation of our PCR products were done on March 1.

Joseph Silo 13:00, 26 February 2011 (EST)

Completed the Zymo Gel Purification step on sbb1120, sbb1131, and sbb1105. Eluted each with 8 microliters of water.

Joseph Silo 12:55, 22 February 2011 (EST)

Dr. Anderson redid all of the PCR products because people may have used the wrong buffer during the Regular Zymo Cleanup step. Did an EcoRI/BamHI Digest of PCR Products sbb1120, sbb1131, and sbb1105. Ran an agarose gel (Gel C with sbb1120, sbb1131, and sbb1105 in C7, C8, and C6, respectively).

Joseph Silo 14:25, 17 February 2011 (EST)

Did an analytical gel for PCR parts sbb1120, sbb1131, and sbb1105. PCR parts are in Analytical Gel Pic 3, in lanes 8, 9, 10, respectively. See image below.

Bands are good.

Did a Regular Zymo Cleanup:

Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.

Transfer into the Zymo column (small clear guys)

Spin through, discard waste.

Add 200 uL of A4 Wash Buffer (which is basically 70% ethanol)

Spin through, discard waste.

Add 200 uL of A4 Wash Buffer

Spin through, discard waste.

Spin for 90 seconds, full speed to dry.

Elute with water into a fresh Eppendorf tube, use the same volume of water (8μL) as the volume of the original reaction
That is, I eluted with 33 microliters of water.

Joseph Silo 14:10, 15 February 2011 (EST)

PCR reactions were set up for parts sbb1120, sbb1131, and sbb1105 using MG1655 genomic DNA as templates. The "Cloning by PCR" protocol was used.

Joseph Silo 23:49, 14 February 2011 (EST)

I'm ready.