Navigate to the Previous or the Next Entry

Contents 1 lab 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 USER ligation and transformation 1.3.2 PCR for Biobrick parts 1.3.3 Gel purification 1.3.4 colony PCR to verify AMO insert 1.4 Results 1.4.1 nanodrop measurement of ara spl plasmids 1.5 Conclusion 2 lab 115 2.1 Main purpose 2.2 Who was in the lab 2.3 Procedure 2.4 Results 2.4.1 Colorimetric results 2.4.2 N2O measurement 2.5 Conclusion

lab 208

---

Main purpose

---

• USER reaction
• PCR for biobrick parts
• gel purification of pZA21::ara with USER endings
• colony PCR

Who was in the lab

---

Kristian, Henrike

Procedure

---

USER ligation and transformation

Redid for HAO and cyc in arabinose inducible pZA21::araBAD and for Nir fragments

PCR for Biobrick parts

Set up a new PCR reaction for Biobrick parts using HF buffer and 5% DMSO but no MgCl2. PCR was run on a touchdown program

primers: 53a, 53b

template: Sec2 miniprep

program:

temperature	time	cycles
98C	2:00	-
98C	0:10	10
63C	1:00	10
-0.5C per cycle
72C	1:00	10
98C	0:10	25
53C	1:00	25
72C	1:00	25
72C	5:00	-
10C	hold	-

Gel purification

Gel purified the linearized plasmid pZA21::ara with USER endings

colony PCR to verify AMO insert

Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template

primers: 53a, 53b

program:

temperature	time	cycles
98C	10:00	-
98C	0:10	36
56C	0:30	36
72C	2:00	36
72C	5:00	-
10C	hold	-

Results

---

nanodrop measurement of ara spl plasmids

Conclusion

---

Purification of plasmids containing the araBAD SPL and RFP successful but with varying concentration.

lab 115

---

Main purpose

---

Run Experiment 2 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

time	nitrite (mg/L)	nitrate (mg/L)	ammonium (mg/L)
0	<0.02	<1	3.8
5	<0.02	<1	<2
10	<0.02	<1	0.3
spike	0.7	<1	<2
15	0.7	<1	0.7
20	0.71	<1	0.2
25	0.72	<1	<2
second spike	1.46	1.7	2.7
35	1.42	1.6	<2

For Sample B

time	nitrite (mg/L)	nitrate (mg/L)	ammonium (mg/L)
0	<0.02	-	<2
5	<0.02	-	<2
10	<0.02	-	<2
spike	0.36	-	<2
15	0.4	-	<2
20	0.4	<1	<2
second spike	2.12	2.3	<2
30	1.78	2.6	0.4
35	1.74	2.7	<2

N₂O measurement

Conclusion

There was no response from native E.coli.

Navigate to the Previous or the Next Entry