IGEM:MIT/2007/Notebook/2007-8-10

Agenda

 * 1) Run Western Blot (at Belcher Lab with Rana and Robbie)

Preparing Samples/Inducing

 * 1. Grow Overnight
 * 2. Dilute to OD 0.15 and Induce
 * 3. Incubate @ 37C on Shaker for 1 hour until grown to OD of approximately 0.4
 * 4. Spin down 1 mL
 * 5. Resuspend in 20µl water
 * 6. Add Loading Buffer
 * 7. Heat
 * 8. Load Gel
 * 9. Run for 30 minutes @ 400V

Transfer

 * Order of layers: 2 pieces padding, filter paper, gel, transfer membrane, filter paper, 2 pieces padding
 * Run for 1 hour @ 30V

Blocking/ Antibodies

 * 1. Block for 1 hour on shaker in 0.5% Milk in 0.1% TBS+Tween
 * 2. Wash 3 times for 5 minutes in TBS+Tween
 * 3. Shake for 1 hour in primary T7 Mouse Monoclonal Antibody (1:10,000 dilution, 40ng/mL) diluted in Blocking Buffer
 * 4. Wash 5 times
 * 5. Shake for 1 hour in secondary Goat Anti-Mouse Antibody (1:2,000 dilution, 0.5ng/mL) diluted in Blocking Buffer
 * 6. Wash 5 times

Imaging

 * 1. Dip in substrate
 * 2. Image using Kodak program
 * 3. Take image without fluorescence first to record ladder position
 * 4. Preview and calculate necessary exposure time
 * 5. Image and save to desktop (email)

Western Blot
Lanes: 1 Invitrogen BenchMark Ladder 2 T  1E-5 3 T  1E-7 4 T  1E-8 5 T  0 6 U  1E-5 7 U  1E-8 8 U  0 9 Positive Control

Key:

T = DH5a transformed with F2620.B0034.CPX in plasmid pSB1AC3

U = DH5a untransformed

Numbers = concentration of AHL added to culture to induce transcription



There are four extra bands in lanes with transformed cells. Zero bands in non-transformed cells. Extra bands may be from: ampR, cmR...? Will insert a transcriptional stop via Standard Assembly.