Team:Aachen/Notebook/Protocols/detection

From 2014.igem.org

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(→2D Detection of IPTG and HSL)
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== Chip Production ==
== Chip Production ==
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=== Cell Preparation ===
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'''Cell Preparation'''
# over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
# over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
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# re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .
# re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .
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'''Agar Preparation'''
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=== Agar Preparation ===
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# autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
# autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
# cool it down to 45 °C in a water bath.
# cool it down to 45 °C in a water bath.
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'''Chip Preparation'''
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=== Chip Preparation ===
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# mix the cooled medium with the cells by inverting gently.
# mix the cooled medium with the cells by inverting gently.
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# put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
# put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
# incubate two chips for 1 h at 37 °C prior to induction.
# incubate two chips for 1 h at 37 °C prior to induction.
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Revision as of 15:24, 16 October 2014

2D Detection of IPTG and HSL

The protocol for the chip production was developed by this year's iGEM Team Aachen with Anna as driving force and person in charge. A lot of parameters were tested and the final protocol for the production of the sensor chips the way there were used for detection in the project is published below.

Chip Production

Cell Preparation

  1. over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
  2. centrifuge all 50 mL by 3000 g for 10 min at RT (21 °C).
  3. discard the supernatant
  4. re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .

Agar Preparation

  1. autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
  2. cool it down to 45 °C in a water bath.

Chip Preparation

  1. mix the cooled medium with the cells by inverting gently.
  2. pour it in the chip form, avoiding bubble formation (!).
  3. wait for approximately 20 min until the agar has solidified.
  4. cut out the chips with a scalpel.
  5. put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
  6. incubate two chips for 1 h at 37 °C prior to induction.
Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png
Sensor-chip manufacturing
Scheme illustrating the work flow during chip production.

Measurement of Fluorescence

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