Team:Amsterdam/project/protocols/
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<h2>Long time exposure protocol</h2> | <h2>Long time exposure protocol</h2> | ||
- | + | '''Day 1''' | |
- | 1. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial | + | *1. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial |
cultures (with appropriate antibiotic) | cultures (with appropriate antibiotic) | ||
- | 2. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial | + | *2. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial |
cultures (with appropriate antibiotic) | cultures (with appropriate antibiotic) | ||
- | + | '''Day 2''' | |
- | 1. Start of the day: To 3 cultures from Day 1, Step 1 add to each 15 ul of 1M IPTG solution | + | *1. Start of the day: To 3 cultures from Day 1, Step 1 add to each 15 ul of 1M IPTG solution |
- | 2. Start of the day: To 3 cultures from Day 1, Step 2 add to each 120 ul of 100% (1g/ml) | + | *2. Start of the day: To 3 cultures from Day 1, Step 2 add to each 120 ul of 100% (1g/ml) |
Arabinose solution | Arabinose solution | ||
- | 3. Keep all cultures incubated at 37C | + | *3. Keep all cultures incubated at 37C |
- | + | '''Day 3''' | |
- | 1. Start of day: From 1 of the cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for | + | *1. Start of day: From 1 of the cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for |
miniprep 1 for western blot), discard rest of culture (IPTG+ 1 day exposure) | miniprep 1 for western blot), discard rest of culture (IPTG+ 1 day exposure) | ||
- | 2. Start of day: From 1 of the cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for | + | *2. Start of day: From 1 of the cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for |
miniprep 1 for western blot), discard rest of culture (IPTG- 1 day negative control) | miniprep 1 for western blot), discard rest of culture (IPTG- 1 day negative control) | ||
- | 3. Start of day: From 1 of the cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take | + | *3. Start of day: From 1 of the cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take |
2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 1 day | 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 1 day | ||
exposure) | exposure) | ||
- | 4. Start of day: From 1 of the cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take | + | *4. Start of day: From 1 of the cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take |
2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 1 day | 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 1 day | ||
negative control) | negative control) | ||
- | 5. Start of day: To the 2 remaining cultures of Day 2, Step 2 each add 120 ul of 100% (1g/ml) | + | *5. Start of day: To the 2 remaining cultures of Day 2, Step 2 each add 120 ul of 100% (1g/ml) |
Arabinose solution | Arabinose solution | ||
- | 6. Keep all (remaining) cultures incubated at 37C | + | *6. Keep all (remaining) cultures incubated at 37C |
- | + | '''Day 4''' | |
- | 1. Start of day: From 1 of the (remaining) cultures from Day 2, Step 1 take 2x a 1.65ml sample | + | *1. Start of day: From 1 of the (remaining) cultures from Day 2, Step 1 take 2x a 1.65ml sample |
(1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 2days exposure) | (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 2days exposure) | ||
- | 2. Start of day: From 1 of the (remaining) cultures from Day 2, Step 2 take 2x a 1.65ml sample | + | *2. Start of day: From 1 of the (remaining) cultures from Day 2, Step 2 take 2x a 1.65ml sample |
(1 for miniprep 1 for western blot), discard rest of culture (IPTG- 2 days negative control) | (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 2 days negative control) | ||
- | 3. Start of day: From 1 of the (remaining) cultures from Day 1, Step 1 (and not used in Day 2, | + | *3. Start of day: From 1 of the (remaining) cultures from Day 1, Step 1 (and not used in Day 2, |
Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture | Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture | ||
(ARA+ 2 days exposure) | (ARA+ 2 days exposure) | ||
- | 4. Start of day: From 1 of the (remaining) cultures from Day 1, Step 2 (and not used in Day 2, | + | *4. Start of day: From 1 of the (remaining) cultures from Day 1, Step 2 (and not used in Day 2, |
Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture | Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture | ||
(ARA- 2 days negative control) | (ARA- 2 days negative control) | ||
- | + | *5. Start of day: To the 1 remaining culture of Day 2, Step 2 add 120 ul of 100% (1g/ml) | |
- | 5. Start of day: To the 1 remaining culture of Day 2, Step 2 add 120 ul of 100% (1g/ml) | + | |
Arabinose solution | Arabinose solution | ||
- | 6. Keep all (remaining) cultures incubated at 37C | + | *6. Keep all (remaining) cultures incubated at 37C |
- | + | '''Day 5''' | |
- | 1. Start of day: From the 1 remaining culture from Day 2, Step 1 take 2x a 1.65ml sample (1 for | + | *1. Start of day: From the 1 remaining culture from Day 2, Step 1 take 2x a 1.65ml sample (1 for |
miniprep 1 for western blot), discard rest of culture (IPTG+ 3 days exposure) | miniprep 1 for western blot), discard rest of culture (IPTG+ 3 days exposure) | ||
- | 2. Start of day: From the 1 remaining culture from Day 2, Step 2 take 2x a 1.65ml sample (1 for | + | *2. Start of day: From the 1 remaining culture from Day 2, Step 2 take 2x a 1.65ml sample (1 for |
miniprep 1 for western blot), discard rest of culture (IPTG- 3 days negative control) | miniprep 1 for western blot), discard rest of culture (IPTG- 3 days negative control) | ||
- | 3. Start of day: From the 1 remaining culture from Day 1, Step 1 (and not used in Day 2, Step 1) | + | *3. Start of day: From the 1 remaining culture from Day 1, Step 1 (and not used in Day 2, Step 1) |
take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 3 | take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 3 | ||
days exposure) | days exposure) | ||
- | 4. Start of day: From the 1 remaining culture from Day 1, Step 2 (and not used in Day 2, Step 2) | + | *4. Start of day: From the 1 remaining culture from Day 1, Step 2 (and not used in Day 2, Step 2) |
take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 3 | take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 3 | ||
days negative control) | days negative control) |
Revision as of 19:16, 23 September 2012