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From 2012.igem.org

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Revision as of 01:57, 27 September 2012

For WNu cell line which has native secreted nuclease activity:

1. Prepare 11-16 plates (10ml LBAgar+10ul AMP +10 ul 1M IPTG). IPTG induces the lac promoter which in turn activates the transcription of nuclease.

2. Streak cells onto all plates at the same time

3. Incubate at 37°C

4. Apply hydrochloric acid (HCl) to the first plate before putting in the incubator (set time as zero)

5. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours.

6. When the reading is taken, observe the following:

a) Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)

b) Diameter of the halo that is achieved once HCl is applied

c) OD from a)

d) Estimate of the depth of the colony on the agar plate

For BL21 cell line that has been modified to contain nuclease:

1. Prepare 11-16 plates (LB Agar + CMP)

2. Streak cells onto all plates at the same time

3. Incubate at 37°C

4. Apply HCl to the first plate before putting in the incubator (set time as zero)

5. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours.

6. When the reading is taken, observe the following:

a. Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours)

b. Diameter of the halo that is achieved once HCL is applied

c. OD from a)

d. Estimate of the depth of the colony on the agar plate