Team:TU-Munich/Notebook/Labjournal

From 2013.igem.org

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=='''Monday, April 22nd'''==
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=='''Monday, April 29nd'''==
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===  Miniprep of pRK792, pRK793, pRIL, ereA, ereB ===
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* The procedure will have to be repeated for pRIL
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Revision as of 15:32, 29 April 2013

Display:
General
Vector Design
Effectors
Viability Sensor
Kill Switch
Safety mechanism

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You can also click on individual experiments to show/hide them
Jump to:
Week 1 22.4-28.4
Week 2 29.4-05.5
Week 3 06.5-12.5
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100 bp GeneRuler:

PageRuler Plus:

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).

Wednesday, April 24th

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 20130424 PhytochromeB RFC25 AgeI NgoMIV.png

Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)

Investigator: Jeff, Leonie, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with EreA and EreB (pDEST14).

Procedure:

  • Plasmid DNA was received dried in paper from McMaster University.
  • DNA was resuspended in ddH2O
  • 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.
  • The transformated cells 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 on chlorampenicol and ampicillin plates.

Week 2

Monday, April 29nd

Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Investigator: Leonie

Aim of the experiment: Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Procedure:

  • Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
  • Concentrations were determined by measuring the absorption:
Plasmid c [ng/µl]
pRK792 214,5
pRK793 134,3
pRIL 6,1
ereA 183,4
ereB 98,3
  • The procedure will have to be repeated for pRIL

Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Investigator: Andreas, Florian

Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)

Procedure:

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