Team:DTU-Denmark/Notebook/19 August 2013

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{{:Team:DTU-Denmark/Templates/StartPage|19 August 2013}}
{{:Team:DTU-Denmark/Templates/StartPage|19 August 2013}}
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=Lab 208=
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=lab 208=
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<hr/>
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==Main purpose==
==Main purpose==
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*Preparation of constructs to send for sequencing
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*Gradient PCR for creation of Biobricks
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*PCR to linearize pZA21::RFP::araBAD
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*make Nir1 and Nir2 templates for Morten Nørholm
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*check-up PCR of Hello World construct
==Who was in the lab==
==Who was in the lab==
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5uL of 100~150ng/ul plasmid + 5uL of 5uM primers.
5uL of 100~150ng/ul plasmid + 5uL of 5uM primers.
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An overview of the sample composition can be found in the following link:
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An overview of the sample composition can be found in this 
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[[File:Construct for sequencing - Sheet1.pdf]]
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[https://static.igem.org/mediawiki/2013/9/90/Construct_for_sequencing_-_Sheet1.pdf file]
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The samples were sent to Macrogen for sequencing.
The samples were sent to Macrogen for sequencing.
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===PCR on one of the Hello Wolrd constructs to confirm it's intact===
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===PCR on one of the Hello World 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.
Performed PCR on the construct containing Sec2 to check the template is correct because our PCRs on it are not working.
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==Procedure==
==Procedure==
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* Weigh 77.14 mg NaNO<sub>3</sub> and dissolve in 100 ml for 10X solution of nitrate.
* Weigh 77.14 mg NaNO<sub>3</sub> and dissolve in 100 ml for 10X solution of nitrate.
* Weigh 123.2 mg NaNO<sub>2</sub> and dissolve in 50 ml for 1000X solution and then dilute 1 ml in 99 ml for 10X solution
* Weigh 123.2 mg NaNO<sub>2</sub> and dissolve in 50 ml for 1000X solution and then dilute 1 ml in 99 ml for 10X solution
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{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 17:38, 28 September 2013

19 August 2013

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Contents

Lab 208

Main purpose

  • Preparation of constructs to send for sequencing
  • Gradient PCR for creation of Biobricks
  • PCR to linearize pZA21::RFP::araBAD
  • make Nir1 and Nir2 templates for Morten Nørholm
  • check-up PCR of Hello World construct

Who was in the lab

Kristian, Henrike, Julia

Procedure

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 World 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

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

2013-08-19 big gel.jpg


  • 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

2013-08-19 small gel.jpg

Conclusion

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

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

Ariadni, Helen

Procedure

  • 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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