Team:Imperial College London/Wetlab/Protocols/IPTGgrowth

From 2009.igem.org

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(→Things needed)
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2) Add the following volumes of IPTG to each of the labeled wells: <br>
2) Add the following volumes of IPTG to each of the labeled wells: <br>
A 1: 0 ul of IPTG ( 0 uM IPTG)<br>
A 1: 0 ul of IPTG ( 0 uM IPTG)<br>
-
A 3: 1 ul of 0.01M IPTG (50 uM IPTG )<br>
+
A 2: 1 ul of 0.01M IPTG (50 uM IPTG )<br>
-
A 4: 2ul of 0.01M IPTG (100 uM IPTG)<br>
+
A 3: 2ul of 0.01M IPTG (100 uM IPTG)<br>
-
A 5: 0.5ul of 0.1M IPTG (250 uM IPTG)<br>
+
A 4: 0.5ul of 0.1M IPTG (250 uM IPTG)<br>
-
A 6: 1.0ul of 0.1M IPTG (500 uM IPTG)<br>
+
A 5: 1.0ul of 0.1M IPTG (500 uM IPTG)<br>
 +
A 6: 1.5ul of 0.1M IPTG (750 uM IPTG)<br>
A 7: 2.0ul of 0.1M IPTG (1.0 mM IPTG)<br>
A 7: 2.0ul of 0.1M IPTG (1.0 mM IPTG)<br>
A 8: 0.5ul of 1M IPTG (2.5 mM IPTG)<br>
A 8: 0.5ul of 1M IPTG (2.5 mM IPTG)<br>
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A10: Blank <br>
A10: Blank <br>
<br>
<br>
-
Perform 3 replicates<br>
+
Perform 2 replicates<br>
Ie. A1 to A10<br>
Ie. A1 to A10<br>
Up till C10<br>
Up till C10<br>

Revision as of 09:56, 12 September 2009

Contents

IPTG effect on growth

Aims

  • Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
  • Determine the effect of IPTG toxicity on growth w/o any protein production complications

Assay

Normal cells (without any constructs) will be grown on M9 media until OD=0.7.
IPTG of various concentrations will then be added, and the OD of the cells will be followed over time

Equipment

  • Multi-plate reader
  • 15ml falcon tubes
  • 96 well plates

Reagents

  • M9 Minimal Media with 0.2% Casamino acids, 0.5% Glucose and Strep

(for M9 minimal media preparation, refer to Secondary Carbon Sources)

  • LB Media

Protocol

Day 1

Things needed

  • LB + Strep

Inoculation of cells

1) Using a loop, pick out a single colony of Top-10 cells from a Top-10 Strep plate (in cold room). Innoculate the cells on LB broth (with Strep) and grow them overnight at 37 °C with spinning.

Day 2

1) Before noon, check that the starter culture of Top-10 in LB has grown (ie. Turned cloudy). Take out and transfer to cold room

2) At 3pm, dilute cells 1:50 (cells: media) into 10ml of supplemented M9 media. Grow them overnight at 37 °C

Day 3

Things needed

  • Multi-well plate reader
  • 96 well plate
  • IPTG
  • supplemented M9 minimal media
  • 15ml falcon tubes
  • Eppendorf tubes

Preparing IPTG stock solutions

1) Take out an IPTG 1M aliquot from -20°C Freezer and wait to thaw


2) Create 2 diluted solutions in 2 eppendorf tubes:

0.1M IPTG- add 100ul of 1M IPTG to 900ul of sterile H2O

0.01M IPTG- add 10ul of 1M IPTG to 1ml of sterile H2O

Mix well.

Growing up cells

1) Dilute the cells in fresh M9 media (5ml)
2)The OD is monitered by transferring 200ul aliquots into a 96 well plate and measuring the absorbance at 600nm.

IPTG experiment

Prepare a 96 well plate:
1) After the OD reaches 0.7, add in 200ul of Top-10 cells (from day 2) to wells A1 to A9.

2) Add the following volumes of IPTG to each of the labeled wells:
A 1: 0 ul of IPTG ( 0 uM IPTG)
A 2: 1 ul of 0.01M IPTG (50 uM IPTG )
A 3: 2ul of 0.01M IPTG (100 uM IPTG)
A 4: 0.5ul of 0.1M IPTG (250 uM IPTG)
A 5: 1.0ul of 0.1M IPTG (500 uM IPTG)
A 6: 1.5ul of 0.1M IPTG (750 uM IPTG)
A 7: 2.0ul of 0.1M IPTG (1.0 mM IPTG)
A 8: 0.5ul of 1M IPTG (2.5 mM IPTG)
A 9: 1.0ul of 1M IPTG (5.0 mM IPTG)
A10: Blank

Perform 2 replicates
Ie. A1 to A10
Up till C10
Mix well.

3) To blank well, add in 200ul of M9+ 0.5% Glucose

4) Set up the plate reader to take OD readings at 600nm every half hour. (Using script IGEM Abs). Obtain readings overnight. Note: readings after 8 hours might not longer be accurate due to evaporation.

5)Determine background absorbance by measuring control well. This should be subtracted from subsequent absorbance readings.

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