IGEM:MIT/2007/Notebook/2007-8-10: Difference between revisions
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==Agenda== | ==Agenda== | ||
#Run Western Blot (at Belcher Lab with Rana and Robbie) | #Run Western Blot (at Belcher Lab with Rana and Robbie) | ||
==Updated Western Blot Protocol== | |||
===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== | ==Western Blot== |
Revision as of 14:58, 11 August 2007
Agenda
- Run Western Blot (at Belcher Lab with Rana and Robbie)
Updated Western Blot Protocol
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