Team:Amsterdam/project/protocols/
From 2012.igem.org
(Difference between revisions)
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Notes: | Notes: | ||
*Keep buffers, tips, tubes, rotors, etc. ice cold<br/> | *Keep buffers, tips, tubes, rotors, etc. ice cold<br/> | ||
+ | |||
+ | <h2>Long time exposure protocol</h2> | ||
+ | <h4>Day 1</h4> | ||
+ | |||
+ | 1. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial | ||
+ | cultures (with appropriate antibiotic) | ||
+ | 2. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial | ||
+ | cultures (with appropriate antibiotic) | ||
+ | |||
+ | <h4>Day 2</h4> | ||
+ | |||
+ | 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) | ||
+ | Arabinose solution | ||
+ | 3. Keep all cultures incubated at 37C | ||
+ | |||
+ | <h4>Day 3</h4> | ||
+ | |||
+ | 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) | ||
+ | 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) | ||
+ | 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 | ||
+ | exposure) | ||
+ | 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 | ||
+ | negative control) | ||
+ | 5. Start of day: To the 2 remaining cultures of Day 2, Step 2 each add 120 ul of 100% (1g/ml) | ||
+ | Arabinose solution | ||
+ | 6. Keep all (remaining) cultures incubated at 37C | ||
+ | |||
+ | <h4>Day 4</h4> | ||
+ | |||
+ | 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) | ||
+ | 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) | ||
+ | 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 | ||
+ | (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, | ||
+ | Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture | ||
+ | (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) | ||
+ | Arabinose solution | ||
+ | 6. Keep all (remaining) cultures incubated at 37C | ||
+ | |||
+ | <h4>Day 5</h4> | ||
+ | |||
+ | 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) | ||
+ | 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) | ||
+ | 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 | ||
+ | days exposure) | ||
+ | 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 | ||
+ | days negative control) | ||
</div> | </div> |
Revision as of 19:14, 23 September 2012