IGEM:MIT/2007/Notebook/2007-7-23

=Agenda=
 * 1) Polystyrene Assay
 * 2) *Morning: get correct OD
 * 3) *Add AHL
 * 4) *Afternoon: Test
 * 5) Digest I13500
 * 6) Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids)
 * 7) *Run overnight
 * 8) Dilute sequencing primers
 * 9) *Sequence PHIE6P
 * 10) *Sequence F2620+B0034+CPX
 * 11) Order T7 antibody

Digestion of I13500

 * Made two mixtures

4.5 µl I13500 (Miniprep #1, 223.5 ng/µl) 1 µl  XbaI 1 µl  PstI 0.5 µl BSA 5 µl  NEB3 Buffer 38 µl H20 - 50 µl Total
 * Placed in 37C @ 10:20am --> Take out @ 1:20pm

PCR Purification of Digested I13500 (JCH)

 * Nanodrop: 23.3 ng/µL

3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3 (SK, JH, BH)
Part         Length          Nanodrop pSB1AT3      3450 bp         125 ng/µl pT040        700             29.9 I13500       740             23.3 Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3 Insert 1: 29.9x = (4)(700/3450)(50) --> x = 1.357 µl of Mer Insert 2: 23.3x = (4)(740/3450)(50) --> x = 1.841 µl of I13500 (cut with X and P) Positive Sample, "iGEM 7/23 Lig + mer+I13500 in 1AT3"
 * Ligation Mixtures Calculations (see wiki Notebook 7/9)
 * Ligation Mixtures

0.4 µl pSB1AT3 1.36 µl pT040 (Mer) 1.84 µl I13500 (RBS+GFP) 0.5 µl ligase (T4) 2.0 µl ligase buffer 10x (T4) 13.9 µl H2O -- 20.0 µl total

Negative Control (backbone only), "iGEM 7/23 Lig. - in 1AT3" 0.4 µl pSB1AT3 0.5 µl ligase (T4) 2.0 µl ligase buffer 10x 17.1 µl H2O --- 20.0 µl total


 * Two PCR tubes (+ and -) put in Thermocycler, Block A, OVERNIGHT -- started at 3 PM
 * Program name "16IGEM"
 * No heated lid
 * 16 degrees Celsius, forever

Diluted BioBrick Sequencing Primers
You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room).

Diluted from 100 µM stock to 32.2 µM with total volume of 1.55 mL each.

Made 1 VF2 and 1 VR for iGEM use.

Polystyrene Assay Protocol

 * Cell Lines:
 * Controls: DH5-alpha (not transformed)
 * FHUA PHIE6 --> IPTG
 * CPX --> AHL
 * Media:
 * H20
 * PBS
 * PBS + Tween + BSA


 * Protocol
 * 1) Add AHL/IPTG to Liquid Culture
 * 2) Wait an hour for AHL induced expression
 * 3) Incubate wells in wash media for 30 minutes on rocker at speed 5
 * 4) Centrifuge cells out of Liquid Culture media
 * 5) Resuspend in wash media
 * 6) Add cells to appropriate well
 * 7) Place on rocker for 1 hour at speed 5
 * 8) Aspirate/Pipet out media in wells
 * 9) Resuspend in 150 ul media and wash for 15 minutes on rocker (repeat 2-3 times)