Team:DTU-Denmark/Notebook/8 July 2013

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=208=
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{{:Team:DTU-Denmark/Templates/StartPage|8 July 2013}}
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=Lab 208=
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<hr/>
==Main purpose==
==Main purpose==
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Run gel with 1 AMO, 10 AMO, 20 AMO genes from "Nitrosomonas Europaea".
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<hr/>
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Purification and extraction of the gel for 10 AMO.  
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Run gel with 1 uL AMO, 10 uL AMO, 20 uL AMO genes from ''Nitrosomonas europaea''.
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Run 2nd gel with all the sample of 10 AMO.
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Purification and extraction of the gel for 10 uL AMO.  
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Run gel with HAO and CYC genes "Nitrosomonas Europaea".
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Run 2nd gel with all the sample of 10 uL AMO.
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Run gel with HAO and CYC genes ''Nitrosomonas europaea''.
==Who was in the lab==
==Who was in the lab==
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<hr/>
Kristian, Ariadni, Gosia
Kristian, Ariadni, Gosia
==Procedure==
==Procedure==
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<hr/>
Run 1st gel (AMO):  
Run 1st gel (AMO):  
* 1ul of dye
* 1ul of dye
Line 24: Line 30:
Purification of second gel according to the same protocol.
Purification of second gel according to the same protocol.
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Measure the concentration of the purified AMO using Nanodrop (pouring together both samples.
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Measure the concentration of the purified AMO using Nanodrop (pouring together both samples).
Run 3rd gel (HAO,CYC):  
Run 3rd gel (HAO,CYC):  
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==Results==
==Results==
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Nanodrop measurement:
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<hr/>
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===Nanodrop measurement:===
* 30.4 ng/ul (AMO)
* 30.4 ng/ul (AMO)
* 14.4 ng/ul (HAO)
* 14.4 ng/ul (HAO)
* 11.7 ng/ul (CYC)
* 11.7 ng/ul (CYC)
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===Pictures from gels===
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Gel AMO
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Wells:
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*1: 1 AMO
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*2: 1 AMO
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*3: 10 AMO
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*4: 10 AMO
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*5: 20 AMO
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*6: 20 AMO
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*7: 1 Kb Plus DNA Ladder
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[[File:2013-07-08_AMO colony PCR.jpg | 600px]]
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==Conclusions==
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<hr/>
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===Conclusion for AMO===
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The bands for 10 AMO are thick and it is not visible how many base-pairs they have, so gel extraction and purification is needed.
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Gel HAO-CYC
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Wells:
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*1: 1 Kb Plus DNA Ladder
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*2: 3 HAO
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*3: 6 HAO
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*4: 10 HAO
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*5: 3 HAO
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*6: 6 HAO
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*7: 10 HAO
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Around 3000 base-pairs
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*8: 3 CYC
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*9: 6 CYC
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*10: 10 CYC
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*11: 3 CYC
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*12: 6 CYC
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*13: 10 CYC
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Around 1650 base-pairs
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*14: 1 Kb Plus DNA Ladder
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[[File:2013-07-08_HAO_CYC_2.jpg | 600px]]
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===Conclusion for HAO and CYC===
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The bands have shown that the results are close to 2866 bp for HAO and close to 1497 bp for CYC.
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Gel with purified AMO,HAO and CYC
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Wells
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*8: 1 Kb Plus DNA Ladder
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*11: purified AMO
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(Around 3141 bps)
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*12: purified HAO
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(Around 2866 bps)
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*13: purified CYC
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(Around 1467 bps)
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[[File:2013-07-09_nir_PCR_product..jpg |600px]]
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===Conclusion for Nir===
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We clearly see band - not the best but not the worst either. The band are over 12kb according to the ladder which does not match up with the around 9102 we expected from the primer design. We rand a blast on the primers to see if there where other possible binding sites in the chromosome. No good match was found so we purified and will run the purification on a new gel.
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{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 20:39, 16 September 2013

8 July 2013

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Contents

Lab 208

Main purpose

Run gel with 1 uL AMO, 10 uL AMO, 20 uL AMO genes from Nitrosomonas europaea. Purification and extraction of the gel for 10 uL AMO. Run 2nd gel with all the sample of 10 uL AMO. Run gel with HAO and CYC genes Nitrosomonas europaea.

Who was in the lab

Kristian, Ariadni, Gosia

Procedure

Run 1st gel (AMO):

  • 1ul of dye
  • 5ul of sample
  • 5ul Kb ladder

Purification of first gel for 10 AMO according to the protocol QIA gel extraction protocol. (given in the Kit)

Run 2nd gel (AMO):

  • 9ul of dye
  • around 20 ul of each sample
  • 12 ul of ladder

Purification of second gel according to the same protocol.

Measure the concentration of the purified AMO using Nanodrop (pouring together both samples).

Run 3rd gel (HAO,CYC):

  • 1ul of dye
  • 5ul of sample
  • 5ul Kb ladder

Purification of DNA of both HAO and CYC.

Results

Nanodrop measurement:

  • 30.4 ng/ul (AMO)
  • 14.4 ng/ul (HAO)
  • 11.7 ng/ul (CYC)

Pictures from gels

Gel AMO

Wells:

  • 1: 1 AMO
  • 2: 1 AMO
  • 3: 10 AMO
  • 4: 10 AMO
  • 5: 20 AMO
  • 6: 20 AMO
  • 7: 1 Kb Plus DNA Ladder

2013-07-08 AMO colony PCR.jpg

Conclusions

Conclusion for AMO

The bands for 10 AMO are thick and it is not visible how many base-pairs they have, so gel extraction and purification is needed.


Gel HAO-CYC

Wells:

  • 1: 1 Kb Plus DNA Ladder
  • 2: 3 HAO
  • 3: 6 HAO
  • 4: 10 HAO
  • 5: 3 HAO
  • 6: 6 HAO
  • 7: 10 HAO

Around 3000 base-pairs

  • 8: 3 CYC
  • 9: 6 CYC
  • 10: 10 CYC
  • 11: 3 CYC
  • 12: 6 CYC
  • 13: 10 CYC

Around 1650 base-pairs

  • 14: 1 Kb Plus DNA Ladder

2013-07-08 HAO CYC 2.jpg

Conclusion for HAO and CYC

The bands have shown that the results are close to 2866 bp for HAO and close to 1497 bp for CYC.


Gel with purified AMO,HAO and CYC

Wells

  • 8: 1 Kb Plus DNA Ladder
  • 11: purified AMO

(Around 3141 bps)

  • 12: purified HAO

(Around 2866 bps)

  • 13: purified CYC

(Around 1467 bps)

2013-07-09 nir PCR product..jpg

Conclusion for Nir

We clearly see band - not the best but not the worst either. The band are over 12kb according to the ladder which does not match up with the around 9102 we expected from the primer design. We rand a blast on the primers to see if there where other possible binding sites in the chromosome. No good match was found so we purified and will run the purification on a new gel.


Navigate to the Previous or the Next Entry

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