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== | ||
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[[Image:westernblotCPX.jpg|thumb|left|400px|''Figure 1''': Western Blot of DH5a expressing CPX]][[Image:Newproject5.jpg|thumb|center|400px|''Figure 2''': Western Blot of DH5a expressing CPX]] | [[Image:westernblotCPX.jpg|thumb|left|400px|''Figure 1''': Western Blot of DH5a expressing CPX]][[Image:Newproject5.jpg|thumb|center|400px|''Figure 2''': Western Blot of DH5a expressing CPX]] | ||
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. |
Latest revision as of 07:32, 13 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
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.