February 16, 2012
Started working on creating parts as per the design specs and construction files.
Worked first on cloning the PhoA oligos. Followed steps on "Cloning by PCR" page. Performed dilutions: 401uL for PhoA-F, 284 for PhoA-R. Put the final product on ice after mixing.
Proceeded to set up PCA on oligos 01/011/02 for the leucine zipper. Followed the PCA steps on the protocol pages (PCA Gene Synthesis) and let sit on ice.
February 17, 2012
Continued working on the Leucine Zipper PCA. Ran a Smal Fragment Zymo Cleanup on pca1. Then proceeded to run the amplification step of PCA on o1/o2/pca1 oligos. Made dilutions of o1/o2 before proceeding. However, I made a big boo boo and added ALL of the pca1, instead of 1uL (like the other oligos). My final product is thus volumous. Currently sitting on ice.
As for the gel, Don't have enough time to finish today. Leaving until next time.
February 21, 2012
Ran another zymo cleanup of the Leucine Zipper. This time I prepared pca2 for digestion, incubating it in the thermocycler for 1 hour. On the PhoA side of things, combined PhoA with loading buffer and ran a gel analysis to ensure that my part was the right size. Continued to set up the PhoA part for digestion as well, incubating it with restriction enzymes and NEB buffer. After an hour of incubation, added agarose buffer and stored on ice.
February 23, 2012
Ran a digest of my PCR Product. My band on the gel ended up being pretty light, so it was difficult to cut all the agarose out. I probably had a little more agarose than I should have. Here is a photo of the gel. My part was in well 7, and extremely light.
Afterwards, I ran a zymo gel cleanup on my digest, heating it and washing/eluting appropriately. I decided to put off LZ ligation until next time, pushing back the ligation of both PhoA and the zipper .
February 24, 2012
Ligated my PCR Product and LZ, but didn't have time to proceed and finish transformation. Scrapped all work from today.
February 27, 2012
Ligated PhoA and Leucine zipper with corresponding vectors. After allowing them to incubate, I prepared the a cell mixture by adding KCM and water. Mixing the cells with the pcr/lz products, I then heatshocked and incubated the products for an hour. Plated the cells afterwards.
February 29, 2012
Completed a miniprep of our the cells. Zach picked our colonies.
March 1, 2012
Ran an analytical gel on phoA-1, phoA-2, and LZ2 products from cell. See Gel 2 from March 1.
March 6, 2012
Unfortunately I forgot which lanes my gel was run in, so I reran an analytical gel on PhoA-1 and PhoA-2. Added 5 ul of each with 2 ul of loading dye and loaded into wells 9 and 10.
March 15, 2012
Analyzed the gel that my products were run on. It seems like the parts are around 4.5 kb, which is around where my products should be. There are some extra bands, but after some discussion, I realized they could be results of partial ligation. Submitted my samples for sequencing.
March 20, 2012
Did sequencing analysis on completed parts with team.
April 3, 2012
Brainstormed protocol for quantifying dimerization transduction in toxr-peptide parts.