Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

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[[File:TUM13_20130423_RFP_Generator_RFC25_AgeI_NgoMIV.png|500px]]
[[File:TUM13_20130423_RFP_Generator_RFC25_AgeI_NgoMIV.png|500px]]
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<!--- this closes the week -->
 
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Sequencing batch were prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (50 - 100&nbsp;ng) and 2&nbsp;µl sequencing primer).
Sequencing batch were prepared after manufacturer's protocol. (15&nbsp;µl of plasmid DNA (50 - 100&nbsp;ng) and 2&nbsp;µl sequencing primer).
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</div>
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=='''Monday, April 22nd'''==
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<div class="safety_mechanism">
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===  Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)  ===
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'''Investigator: Jeff, Leonie, Florian'''
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 +
'''Aim of the experiment:''' Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).
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'''Procedure:'''
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* Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
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</div>
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<div class="safety_mechanism">
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=== Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10 ===
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'''Investigator: Jeff, Leonie, Florian'''
 +
 +
'''Aim of the experiment:''' Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.
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'''Procedure:'''
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* Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
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{|cellspacing="0" border="1"
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|'''volume'''
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|'''reagent'''
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|-
 +
|2.5&nbsp;µl
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|Plasmid DNA P7
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|-
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|2&nbsp;µl
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|NEBuffer 4 (10x)
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|-
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|0.25&nbsp;µl
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|NgoMIV (10&nbsp;U/µl)
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|-
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|0.25&nbsp;µl
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|AgeI-HF (20&nbsp;U/µl)
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|-
 +
|15&nbsp;µl
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|ddH2O
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|-
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|=20&nbsp;µl
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|'''TOTAL'''
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|}
 +
 +
* Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5&nbsp;µl
 +
|Plasmid DNA P8
 +
|-
 +
|2&nbsp;µl
 +
|NEBuffer 4 (10x)
 +
|-
 +
|0.25&nbsp;µl
 +
|NgoMIV (10&nbsp;U/µl)
 +
|-
 +
|0.25&nbsp;µl
 +
|AgeI-HF (20&nbsp;U/µl)
 +
|-
 +
|15&nbsp;µl
 +
|ddH2O
 +
|-
 +
|=20&nbsp;µl
 +
|'''TOTAL'''
 +
|}
 +
 +
* Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5&nbsp;µl
 +
|Plasmid DNA P9
 +
|-
 +
|2&nbsp;µl
 +
|NEBuffer 4 (10x)
 +
|-
 +
|0.25&nbsp;µl
 +
|NgoMIV (10&nbsp;U/µl)
 +
|-
 +
|0.25&nbsp;µl
 +
|AgeI-HF (20&nbsp;U/µl)
 +
|-
 +
|15&nbsp;µl
 +
|ddH2O
 +
|-
 +
|=20&nbsp;µl
 +
|'''TOTAL'''
 +
|}
 +
 +
* Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
 +
{|cellspacing="0" border="1"
 +
|'''volume'''
 +
|'''reagent'''
 +
|-
 +
|2.5&nbsp;µl
 +
|Plasmid DNA P10
 +
|-
 +
|2&nbsp;µl
 +
|NEBuffer 4 (10x)
 +
|-
 +
|0.25&nbsp;µl
 +
|NgoMIV (10&nbsp;U/µl)
 +
|-
 +
|0.25&nbsp;µl
 +
|AgeI-HF (20&nbsp;U/µl)
 +
|-
 +
|15&nbsp;µl
 +
|ddH2O
 +
|-
 +
|=20&nbsp;µl
 +
|'''TOTAL'''
 +
|}
 +
 +
* Incubation for 90&nbsp;min at 37&nbsp;°C.
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 +
* Analytical gelelectrophoresis was performed at 90&nbsp;V for 60&nbsp;min.
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 +
'''Results:'''
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 +
{|cellspacing="0" border="1"
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|1&nbsp;kbp ladder DNA ladder
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|'''P7'''
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|'''P8'''
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|'''P9'''
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|'''P10'''
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|-
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|
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|'''Part is correct'''
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|'''Part is correct'''
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|'''Part is correct'''
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|'''Part is correct'''
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|}
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[[File:TUM13_20130423_RFP_Generator_RFC25_AgeI_NgoMIV.png|500px]]
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</div>
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 +
<!--- this closes the week -->
 +
</div>
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 +
<!--- this closes the labbook-->
</div>
</div>

Revision as of 13:15, 24 April 2013

Display:
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Vector Design
Effectors
Viability Sensor
Kill Switch
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You can also click on individual experiments to show/hide them
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Week 1 22.4-28.4
Week 2 29.4-05.5
Week 3 06.5-12.5
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Labjournal

Week 1

Monday, April 22nd

Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Leonie, Rosario

Aim of the experiment: Transformation of Phytochrome B for protein fusion.

Procedure:

  • CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
  • 2 µl of DNA was added to 100 µl of competent cells and gently mixed.
  • 30 min incubation on ice
  • 5 min. heat shock at 37 °C
  • Adding of 1 ml LB-medium to each tube.
  • Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
  • 100 µl of the cell suspension was plated on one chloramphenicol plate.
  • The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
  • The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Investigator: Jeff, Leonie, Rosario

Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Sequencing of RFP-Generator (RFC25, pSB1C3)

Investigator: Jeff, Leonie, Rosario

Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)

Tuesday, April 23rd

Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Leonie, Rosario, Florian

Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Procedure:

  • pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.
  • Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
  • 4 colonies were picked.
  • These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight

Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)

Investigator: Jeff, Leonie, Rosario, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).


Procedure:

  • Batch for analytical digestion for P4 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P4
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P5
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder P4 P5
Mutation successful Mutation successful!
  • Parts are compliant and do not contain RFC25 forbidden restriction sites.


TUM13 20130423 RFP Generator RFC25 AgeI NgoMIV.png

Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Investigator: Jeff, Leonie, Rosario, Florian

Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2

Procedure:

Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

Monday, April 22nd

Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)

Investigator: Jeff, Leonie, Florian

Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10

Investigator: Jeff, Leonie, Florian

Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.

Procedure:

  • Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P7
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P8
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P9
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
volume reagent
2.5 µl Plasmid DNA P10
2 µl NEBuffer 4 (10x)
0.25 µl NgoMIV (10 U/µl)
0.25 µl AgeI-HF (20 U/µl)
15 µl ddH2O
=20 µl TOTAL
  • Incubation for 90 min at 37 °C.
  • Analytical gelelectrophoresis was performed at 90 V for 60 min.

Results:

1 kbp ladder DNA ladder P7 P8 P9 P10
Part is correct Part is correct Part is correct Part is correct


TUM13 20130423 RFP Generator RFC25 AgeI NgoMIV.png

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