Difference between revisions of "Team:Mingdao/InterLab"
| Line 414: | Line 414: | ||
↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM | ↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM | ||
<p> | <p> | ||
| − | + | ↓ Prepare the serial dilutions of fluorescein as follows: | |
<p> | <p> | ||
<img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt="" | <img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt="" | ||
| Line 421: | Line 421: | ||
↓ Measure fluorescence of all samples in instrument | ↓ Measure fluorescence of all samples in instrument | ||
<p> | <p> | ||
| − | ↓ Record the data | + | ↓ Record the data |
<p> | <p> | ||
Revision as of 01:55, 18 October 2018
Interlab Study
Note
Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study
Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here
Results: the experiment and data presented here were all made by members of team Mingdao
Reference: Fifth International InterLab Measurement Study@iGEM
Instrument
The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader
Introduction
"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."
Goal for the Fifth InterLab
"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work."
"This year, teams participating in the interlab study helped iGEM to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?"
Calibration Reference
Calibration 1:OD600 Reference point - LUDOX Protocol
Materials
1ml LUDOX CL-X
ddH2O
96 well Black Clear Bottom Plate
Method
↓ Add 100 μl LUDOX into wells A1, B1, C1, D1
↓ Add 100 μl of ddH2 O into wells A2,B2,C2,D2
↓ Measure absorbance at 600 nm
↓ Record the data
Result
The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.
Calibration 2: Particle Standard Curve - Microsphere Protocol
Materials
300 μL silica beads Microsphere suspension
ddH2O
96 well Black Clear Bottom Plate
Method
Preparation of the Microsphere stock solution:
↓ Obtain Silica Beads
↓ Pipet 96 μL beads into an eppendorf
↓ Add 904 μL of ddH2O to the microspheres
↓ Vortex well to obtain stock Microsphere Solution.
↓ Preparation of microsphere serial dilutions as follows
↓ Measure Abs 600
↓ Record the data
Result
Raw Data
Particle Standard Curve
Particle Standard Curve(log scale)
Calibration 3: Fluorescence standard curve - Fluorescein Protocol
Materials
Fluorescein (provided in kit)
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
96 well Black Clear Bottom Plate
Method
↓ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. ↓ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM
↓ Prepare the serial dilutions of fluorescein as follows:
↓ Measure fluorescence of all samples in instrument
↓ Record the data
Result
Raw Data
Fluorescein Standard Curves
Fluorescein Standard Curves(log scale)
Cell Measurement
Materials
Competent cells ( Escherichia coli strain DH5 )
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
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes and tips
96 well Black Clear Bottom Plate
Workflow
Method
Day1
↓ Transform Escherichia coli DH5 with these plasmids
Day2
↓ Pick 2 colonies from each group
↓ Inoculate in 5-10 mL LB medium + Cm
↓ Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.
Day 3
↓ Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm
↓ Measure Abs 600 of these 1:10 diluted cultures
↓ Record the data
↓ Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube
↓ Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.
↓ Measure your samples for Abs600 and fluorescence
↓ Record data in your notebook
Result
Fluorescence Raw Reading
Abs600 Raw Reading
Colony Forming Units per E. coli cultures at OD600=0.1
↓ Measure the OD600 of your cell cultures
↓ Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.
↓ Make the following serial dilutions for your triplicates
↓ Aseptically spread plate with 100 μL of the dilutions
↓ Incubate at 37°C overnight
↓ Count colonies after 18-20 hours of growth.
Result
Colony Forming Units per o.1 OD600 E.coli cultures
Quote
“”
Follow us on
Contact us
Phone: +886-4-23341797
EMAIL: biolab@ms.mingdao.edu.tw
No.497, Sec. 1, Zhongshan Rd., Wuri District, Taichung City 414, Taiwan (R.O.C.)