lab 208

Main purpose

Who was in the lab

Procedure

PCR reactions

Set up PCR reactions for:

• Biobrick backbone pSB1C3 with USER endings fitting for our inserts
• constitutive reference promoter in pZA21 (for SPL)
• pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts

first round of PCRs:

pSB1C3:

Four different reaction mixes: HF, HF+5%DMSO, GC, GC+5%DMSO

pZA21::ara for Nir:

Standard reaction mix.

constitutive reference promoter:

Mix with GC, 5%DMSO and 2uL MgCl2.

pSB1C3 and pZA21::ara for Nir were without results (see gel picture). New PCRs were set up with one standard reaction and one reaction with additives. The annealing temperature was lowered.

second round of PCRs:

pSB1C3:

pZA21::ara for Nir:

Additionally set up a PCR for cycAX with USER endings since the amount of fragment is running low.

cycAX for USER:

Standard reaction mix.

Results

Gel

• 1 kb ladder
• pSB1C3 for Biobricks, HF buffer
• pSB1C3 for Biobricks, HF buffer, 5%DMSO
• pSB1C3 for Biobricks, GC buffer
• pSB1C3 for Biobricks, GC buffer, 5%DMSO
• negative pSB1C3 for Biobricks
• pZA21::ara for USER ligation with Nir
• pZA21::ara for USER ligation with Nir (duplicate)
• 1 kb ladder

Note: It seems the ladder overflew.

Conclusion

lab 115

Main purpose

Run Experiment 4 in two different samples anaerobically in order to characterize the behavior of E.coli.

Who was in the lab

Ariadni, Helen

Procedure

Adjusting the temperature at 36 degrees and calibrating the probes as described in Calibration protocol.

Following the protocol Experiment 2

Changing the steps :

6. 4 ml of the overnight culture growing in DM minimal medium

12. The OD was measured OD=0.261 in sample A and OD=0.263 in sample B.

19.

• For sample A after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 2 ml of nitrite solution and continue by taking a sample after spiking. Then we took three more samples and after 15 minutes we spike with 2 ml of nitrite, and finally we took a last sample. The final OD measurement was OD=0.244 and the temperature was 36.8 degrees.

• For sample B after taking the first sample, we continue with two more samples after 5 and 10 minutes. Then we add 1 ml of nitrite solution and continue by taking a sample after spiking. Then we took two more samples and after 10 minutes we spike with 4 ml of nitrite, and then there was a response after 12 minutes were finally we took one sample and after 5 minutes the last. The final OD measurement was OD=0.248 and the temperature was 36.4 degrees.

Results

Colorimetric results

Ranges

• Measuring range 2-75 mg/L NH₄-N
• Measuring range 1-25 mg/L NO₃-N
• Measuring range 0.02-1 mg/L NO₂-N

Standard solutions

• Ammonium - 66.6 mg/L (expected 39 mg/L)
• Nitrite - 0.72 mg/L (expected 0.5 mg/L)
• Nitrate -

For Sample A

For Sample B

Conclusion

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