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Contents 1 Lab 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 Preparation of constructs to send for sequencing 1.3.2 Gradient PCR for creation of Biobricks 1.3.3 PCR to linearize pZA21::RFP::araBAD 1.3.4 PCR on one of the Hello Wolrd constructs to confirm it's intact 1.3.5 PCR on NIR1 and NIR2 via. colony PCR for template to Morten Nørholm 1.4 Results 1.4.1 gels 1.5 Conclusion 2 lab 115 2.1 Main purpose 2.2 Who was in the lab 2.3 Procedure

Lab 208

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Main purpose

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Who was in the lab

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Kristian, Henrike, Julia

Procedure

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Preparation of constructs to send for sequencing

Sec2, TAT3-2, TAT3-1a, CycAX and HAO constructs were prepared for sequencing. Sample preparation was performed following the indications given by the EZ-Seq service by Macrogen:

5uL of 100~150ng/ul plasmid + 5uL of 5uM primers.

An overview of the sample composition can be found in this file

The samples were sent to Macrogen for sequencing.

Gradient PCR for creation of Biobricks

Made a screening to find good conditions for the extraction of our constructs for subsequent ligation into BioBricks.

conditions:

A:

• HF buffer
• 2% DMSO
• 1uL 50mM MgCl2

B:

• HF buffer
• 3% DMSO
• 2uL 50mM MgCl2

C:

• HF buffer
• 5% DMSO
• 1uL 50mM MgCl2

D:

• HF buffer
• 3% DMSO
• 1uL 50mM MgCl2

program:

temperature	time	cycles
98C	2:00	-
98C	0:10	36
annealing temp.	1:00	36
72C	1:00	36
72C	5:00	-
10C	hold	-

The annealing temperature was a gradient from 48C to 63C in 12 steps.

sample #	1	2	3	4	5	6	7	8	9	10	11	12
temperature	48.3	48.7	50.0	51.8	53.3	54.7	56.3	57.5	59.1	60.8	62.5	63

PCR to linearize pZA21::RFP::araBAD

Used GC buffer and 5% DMSO. Made three samples with varying concentrations of 50mM MgCl2: 0,5 uL, 1uL and 2 uL.

template: pZA21::RFP::araBAD

primers: 55a, 55b

program:

temperature	time	cycles
98C	2:00	-
98C	0:10	36
65C	1:00	36
72C	3:00	36
72C	5:00	-
10C	hold	-

PCR on one of the Hello Wolrd constructs to confirm it's intact

Performed PCR on the construct containing Sec2 to check the template is correct because our PCRs on it are not working.

template: Sec2 construct

primers: sequencing primers 4_FW_pZA21 and 5_RV_pZA21

temperature	time	cycles
98C	10:00	-
98C	0:10	36
61C	0:20	36
72C	2:00	36
72C	5:00	-
10C	hold	-

PCR on NIR1 and NIR2 via. colony PCR for template to Morten Nørholm

sample N1 or N2#

HF

HF 2% DMSO 1mL MgCl2 50mM

HF 5% DMSO

HF 1M Betaine

GC

GC 2% DMSO

GC 5% DMSO

GC 5% DMSO 1mL MgCl2 50mM

GC 5% DMSO 2mL MgCl2 50mM

GC 1M Betaine

temperature	time	cycles
98C	10:00	-
98C	0:20	14
71C -0.5C increment	0:30	14
72C	2:30	14
98C	0:20	20
64C	0:30	20
72C	2:30	20
72C	5:00	-
10C	hold	-

Results

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gels

• 1: 1 kb ladder
• 2: N1 HF
• 3: N1 HF 2% DMSO 1 mL MgCl2
• 4: N1 HF 5% DMSO
• 5: N1 HF 1M Betaine
• 6: N1 GC
• 7: N1 GC 2% DMSO
• 8: N1 GC 5% DMSO
• 9: N1 GC 5% DMSO 1 mL MgCL2
• 10: N1 GC 5% DMSO 2 mL MgCl2
• 11: N1 GC 1M Betaine
• 12: N2 HF
• 13: N2 HF 2% DMSO 1 mL MgCl2
• 14: N2 HF 5% DMSO
• 15: N2 HF 1M Betaine
• 16: N2 GC
• 17: N2 GC 2% DMSO
• 18: N2 GC 5% DMSO
• 19: 1 kb ladder

• 1 kb ladder
• N2 GC 5% DMSO 1 mL MgCl2
• N2 GC 5% DMSO 2 mL MgCl2
• N2 GC 1M Betaine
• pZA21::RFP::araBAD 0,5 uL MgCl2
• pZA21::RFP::araBAD 1 uL MgCl2
• pZA21::RFP::araBAD 2 uL MgCl2
• negative pZA21::RFP::araBAD
• check Sec2
• check Sec2 (duplicate)
• negative check Sec2
• screening A1
• screening A2
• screening A3

Conclusion

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The program for Nir colony PCR works as a touchdown PCR and it have a big influence if you add DMSO or Betaine. It seems as 5% DMSO and no additional MgCl2 or 1M Betaine works best and somewhat equally good for both reactions.

lab 115

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Main purpose

Prepare Nitrate and Nitrite standard solutions for colorimetric analysis

• 12 mg/L NO₃-N
• 0.5 mg/L NO₂-N

Who was in the lab

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Ariadni, Helen

Procedure

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• Weigh 77.14 mg NaNO₃ and dissolve in 100 ml for 10X solution of nitrate.
• Weigh 123.2 mg NaNO₂ and dissolve in 50 ml for 1000X solution and then dilute 1 ml in 99 ml for 10X solution

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