Team:UCLA/Notebook/Recombinant Expression/3 August 2015

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AGENDA FOR WEEK 8

Monday:

Move reagents and products over from Boyer to Boelter Hall (chemicals, solutions, plates, gels (used and new), samples, cells, etc.).

Be sure to grab things from the bench, the 4-degree room, and the 4 deg and -20 deg freezers.

Order Interlab Study g-Blocks for all three devices, as well as the reverse primers for the control GFP construct.

Prepare a starter culture of the M2 12-mer in 10ml LB + Chlor

Tuesday:

Lyse the M2 9-mer cell pellets (if the reagents had come in by then) using the protocol listed in Teule, et. al. (Nat. Prot.) -- http://www.nature.com/nprot/journal/v4/n3/full/nprot.2008.250.html

Inoculate a 1L LB + chlor culture with 5 mL of the 12-mer. Induce with IPTG following the above protocol. Take 1mL culture samples of the cells at 1 hr- expression intervals as listen in the protocol.

Wednesday:

Pellet and freeze the 12-mer cells in lysis buffer as following the protocol.

Purify the 9-mer cell lysate using IMAC Batch Method (as previously detailed on the notebook), and save fractions for SDS-PAGE analysis and BCA Assaying.

Pool and concentrate fractions for Material Processing Team to dialyze, lyophilize, and spin using the wet-spin technique.

Optional: Run the 12-mer expression and purification fractions on SDS-PAGE to verify kDA size of samples.

Thursday:

Optional: Lyse the 12-mer cells, boil, and save the lysate for purification in the morning as following the Teule protocol.

The rest of the day will be saved for the SoCal iGEM Meetup.

Friday:

Purify the 12-mer lysate using IMAC Batch Method (as previously detailed on the notebook), and save fractions for SDS-PAGE analysis and BCA Assaying.

Conduct BCA Assay and SDS-PAGE analysis of the 12-mer expression and purification fractions (and the 9-mer fractions, if not done previously in the week).