Difference between revisions of "Team:XJTU-China/InterLab"
RandolphLiu (Talk | contribs) |
|||
| Line 1: | Line 1: | ||
{{XJTU-China}} | {{XJTU-China}} | ||
| − | + | {{XJTU-China/Header}} | |
<html> | <html> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<body> | <body> | ||
<style>.collapse>.panel-body>img{transform: scale(0.8);}</style> | <style>.collapse>.panel-body>img{transform: scale(0.8);}</style> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<div class="container"> | <div class="container"> | ||
<div class="column full_size description"> | <div class="column full_size description"> | ||
| Line 395: | Line 331: | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
</body> | </body> | ||
</html> | </html> | ||
| + | {{XJTU-China/Footer}} | ||
Revision as of 07:01, 14 October 2018
InterLab
Overview
Synthetic biology requires reliable and repeatable measurement. However, the ability to repeat measurements in different labs has been difficult for different units or different ways may be used in various groups. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). In order to improve reproducibility, we used plate readers to take measurements of fluorescence and absorbance this year.
Methods and Materials
Materials
- 1 mL LUDOX (provided in kit)
- ddH2O
- 96 well plate
Methods
- Add 100 μl LUDOX into wells A1, B1, C1, D1
- Add 100 μl of dd2O into wells A2, B2, C2, D2
- Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
Materials
- Fluorescein (provided in kit)
- 10 mL 1xPBS (phosphate buffered saline)
- 96 well plate, black with clear flat bottom
Methods
- Prepare the Microsphere Stock Solution
- Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube
- Add ddH2O to the microspheres
- Vortex well.
- Prepare the Microsphere Stock Solution
- Measure Abs600 of all samples in instrument
Materials:
- Fluorescein (provided in kit)
- 10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline)
- 96 well plate, black with clear flat bottom
Methods:
- Prepare the Fluorescein Stock Solution
- Measure fluorescence of all samples in instrument
Materials
- Competent Cells (Escherichia coli strain DH5α
- LB (Luria Bertani) media
- Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
- 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
- Incubator at 37°C
- Ice bucket with ice
- Micropipettes and tips
- 96 well plate, black with clear flat bottom preferred
- Devices (from Distribution Kit, all in pSB1C3 backbone):
| Device | Part Number | Plate | Location |
| Negative control | BBa_R0040 | Kit Plate 7 | Well 2D |
| Positive control | BBa_l20270 | Kit Plate 7 | Well 2B |
| Test Device 1 | BBa_J364000 | Kit Plate 7 | Well 2F |
| Test Device 2 | BBa_J364001 | Kit Plate 7 | Well 2H |
| Test Device 3 | BBa_J364002 | Kit Plate 7 | Well 2J |
| Test Device 4 | BBa_J364007 | Kit Plate 7 | Well 2L |
| Test Device 5 | BBa_J364008 | Kit Plate 7 | Well 2N |
| Test Device 6 | BBa_J364009 | Kit Plate 7 | Well 2P |
Methods
- Day 1: transform Escherichia coli DH5α with given plasmids (all in pSB1C3)
- Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells
-
Day 3: Cell growth, sampling, and assay
- Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
- Measure Abs 600 of these 1:10 diluted cultures
- Dilute the cultures further to a target Abs600 of 0.02 in 50 mL falcon tube
- Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation.Place the samples on ice.
- Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
- Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
- Measure samples (Abs fluorescence measurement).
Step 1: Starting Sample Preparation
- Measure the OD600 of the cell cultures and dilute to the linear detection range of the plate reader, e.g. to 0.05 – 0.5 OD600 range.
- Dilute the overnight culture to OD600 = 0.1 in 1mL of LB + Cam media. Do this in triplicate for each culture.
- Check the OD600 and make sure it is 0.1.
Step 2: Dilution Series Instructions
For each Starting Sample (total for all 12 showed in italics in paraenthesis):
- We need 3 LB Agar + Cam plates (36 total).
- Prepare three 2.0 mL tubes (36 total) with 1900 ºL of LB + Cam media for Dilutions 1, 2, and 3 (see figure below).
- Prepare two 1.5 mL tubes (24 total) with 900 ºL of LB + Cam media for Dilutions 4 and 5 (see figure below)..
- Label each tube according to the figure below (Dilution 1, etc.) for each Starting Sample.
- Pipet 100 ºL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs.
- Repeat Step 5 for each dilution through to Dilution 5 as shown below.
- Aseptically spead plate 100 ºL on LB + Cam plates for Dilutions 3, 4, and 5.
- Incubate at 37°C overnight and count colonies after 18-20 hours of growth.
Step 3: CFU/mL/OD Calculation Instructions
Based on the assumption that 1 bacterial cell gives rise to 1 colony, colony forming units (CFU) per 1mL of an OD600 = 0.1 culture can be calculated as follows:
- Count the colonies on each plate with fewer than 300 colonies.
- Multiple the colony count by the Final Dilution Factor on each plate.
Calibrations
Fluorescence Raw
Abs600 Raw
CFU counts
