Team:DTU-Denmark/Methods/Protocols
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<br><br> | <br><br> | ||
During our laboratory work several protocols and recipes were applied. To make sure that the work is documented in a systematical and reproducible manner, we have made a list of the procedures and added it to this page. | During our laboratory work several protocols and recipes were applied. To make sure that the work is documented in a systematical and reproducible manner, we have made a list of the procedures and added it to this page. | ||
- | |||
- | |||
<br><br> | <br><br> | ||
<h1>Protocols</h1> | <h1>Protocols</h1> | ||
+ | |||
+ | |||
+ | <h2 id="BioLector" class="protocol-header">Biolector Measurements</h2> | ||
+ | <div class="protocol BioLector"> | ||
+ | Cultures of 2 ml LB were inoculated and incubated at 37 °C and 350 RPM O/N.</br> | ||
+ | The O/N cultures were diluted 15000 folds (first by a 100 dilution in salt water and then by additional 150 folds in LB media). The diluted cultures were distributed in triplicates of 1.5 mL in a BioLector flower plate and were placed in the Biolector. The BioLector was set to incubate at 37 °C and 1000 RPM ensuring optimal mass transfer (oxygen).<br> | ||
+ | The experiment ran for 12.5 hours with measurements of cell density and fluorescence in each well every 5 minutes. The cell density was measured by light scatter at 620 nm and fluorescence was measured at 520 nm with excitation at 488 nm. | ||
+ | </div> | ||
- | <h2 id="Protocol-for-ligation-for-DNA-cloning" class="protocol-header"> | + | <h2 id="Protocol-for-ligation-for-DNA-cloning" class="protocol-header">DNA Ligation</h2> |
<div class="protocol Protocol-for-ligation-for-DNA-cloning"> | <div class="protocol Protocol-for-ligation-for-DNA-cloning"> | ||
Each reaction of 20 μL contained: | Each reaction of 20 μL contained: | ||
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</div> | </div> | ||
+ | |||
+ | <h2 id="FACS" class="protocol-header">FACS Measurements</h2> | ||
+ | <div class="protocol FACS"> | ||
+ | 2 ml LB media was inoculated with a single colony and inoculated at 37 ° and 350 RPM O/N.<br> | ||
+ | The cultures were washed twice in PBS (Phosphate Buffered Saline) and then resuspended in PBS. The suspensions were diluted in PBS to a concentration yielding 1000-1500 events per second. | ||
+ | </div> | ||
+ | |||
- | <h2 id="fluorometer-measurement-protocol" class="protocol-header">Fluorometer | + | <h2 id="fluorometer-measurement-protocol" class="protocol-header">Fluorometer Measurement</h2> |
<div class="protocol fluorometer-measurement-protocol"> | <div class="protocol fluorometer-measurement-protocol"> | ||
<p> | <p> | ||
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- | <h2 id="gel-protocol" class="protocol-header">Gel | + | <h2 id="gel-protocol" class="protocol-header">Gel Electrophoresis </h2> |
<div class="gel-protocol protocol"> | <div class="gel-protocol protocol"> | ||
<p> | <p> | ||
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- | <h2 id="glycerol-stock-protocol" class="protocol-header">Glycerol | + | <h2 id="glycerol-stock-protocol" class="protocol-header">Glycerol Stock-Preservation</h2> |
<div class="protocol glycerol-stock-protocol"> | <div class="protocol glycerol-stock-protocol"> | ||
<ul> | <ul> | ||
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- | <h2 id="IVT-protocol" class="protocol-header"><i>In vitro</i> | + | <h2 id="IVT-protocol" class="protocol-header"><i>In vitro</i> Transcription</h2> |
<div class="protocol IVT-protocol"> | <div class="protocol IVT-protocol"> | ||
To prevent RNase, work in an RNase free environment: | To prevent RNase, work in an RNase free environment: | ||
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<ul> | <ul> | ||
<li>Cells are grown O/N in LB media to OD600 of approximately 4</li> | <li>Cells are grown O/N in LB media to OD600 of approximately 4</li> | ||
- | <li>Cells are washed | + | <li>Cells are washed and resuspended in physiological saline with 200 μM DFHBI-1T</li> |
<li>1 μl cell suspension is placed on a poly-lysin coated microscope slide and covered with a coverslide</li> | <li>1 μl cell suspension is placed on a poly-lysin coated microscope slide and covered with a coverslide</li> | ||
<li>Cells were visualized with 630x magnification using a GFP filter and UV light</li> | <li>Cells were visualized with 630x magnification using a GFP filter and UV light</li> | ||
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<h2 id="PCR-protocol" class="protocol-header">PCR</h2> | <h2 id="PCR-protocol" class="protocol-header">PCR</h2> | ||
<div class="protocol PCR-protocol"> | <div class="protocol PCR-protocol"> | ||
- | <form oninput="MQ.value = parseFloat(reactions.value)*32, HF.value = parseFloat(reactions.value)*10, dNTP.value = parseFloat(reactions.value)*5, pol.value = parseFloat(reactions.value)*0.5, temp.value = parseFloat(reactions.value)*0.5, primer.value = parseFloat(reactions.value)*2"> | + | <form id="PCR-input" oninput="MQ.value = parseFloat(reactions.value)*32, HF.value = parseFloat(reactions.value)*10, dNTP.value = parseFloat(reactions.value)*5, pol.value = parseFloat(reactions.value)*0.5, temp.value = parseFloat(reactions.value)*0.5, primer.value = parseFloat(reactions.value)*2"> |
- | A master mix | + | A master mix was made with final concentration of:</br> |
1X HF buffer</br> | 1X HF buffer</br> | ||
0.2mM dNTP</br> | 0.2mM dNTP</br> | ||
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0.02 units/ml polymerase</br> | 0.02 units/ml polymerase</br> | ||
</br> | </br> | ||
- | The | + | The exact amount of template is not important, and it is very difficult to add too little template. Use our calculator below to calculate the amount of stock solutions to be added to your mastermix. Remember its always a good practice to make mastermix for an additional reaction due to pipetting variations.</br> |
</br> | </br> | ||
<table width="350px" border="0" cellspacing="5" cellpadding="0" style="background-color: transparent;"> | <table width="350px" border="0" cellspacing="5" cellpadding="0" style="background-color: transparent;"> | ||
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</br> | </br> | ||
- | As the polymerase is temperature sensitive it should only be removed from the freezer for short periods of time, therefore add the polymerase as the final part of your mastermix. When the polymerase have been added to keep the mastermix on ice. When the mastermix is complete | + | As the polymerase is temperature sensitive it should only be removed from the freezer for short periods of time, therefore add the polymerase as the final part of your mastermix. When the polymerase have been added to your mastermix, it is important to keep the mastermix on ice. When the mastermix is complete it should be mixed thoroughly by pipetting, not by vortexing since this can ruin the polymerase. The mastermix is aliquoted to individual PCR tubes and the tubes are transferred to a thermo cycler. The annealing temperature should be 5 °C below the melting temperature (Tm) of the primers, this step can be tricky as most Tm predictors tend to disagree. Depending on the polymerase used the elongation time should be 30 seconds per kb of the longest expected fragment. All the primers used in our experimental work, together with their sequences and assigned names, are listed <a href="https://2014.igem.org/Team:DTU-Denmark/Primers">here</a>. |
- | + | ||
</div> | </div> | ||
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- | <h2 id="Plasmid-purification" class="protocol-header">Plasmid | + | <h2 id="Plasmid-purification" class="protocol-header">Plasmid Purification</h2> |
<div class="protocol Plasmid-purification"> | <div class="protocol Plasmid-purification"> | ||
Plasmids were purified from a 2 mL LB culture with the antibiotic that is used as the plasmid’s selection marker, grown at 350 rpm and 37 °C overnight.</br> | Plasmids were purified from a 2 mL LB culture with the antibiotic that is used as the plasmid’s selection marker, grown at 350 rpm and 37 °C overnight.</br> | ||
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- | <h2 id="competent-cells-protocol" class="protocol-header">Preparation of | + | <h2 id="competent-cells-protocol" class="protocol-header">Preparation of Competent Cells</h2> |
<div class="protocol competent-cells-protocol"> | <div class="protocol competent-cells-protocol"> | ||
<ul> | <ul> | ||
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- | <h2 id="restriction-protocol" class="protocol-header">Restriction | + | <h2 id="restriction-protocol" class="protocol-header">Restriction Digestion</h2> |
<div class="protocol restriction-protocol"> | <div class="protocol restriction-protocol"> | ||
<ol> | <ol> | ||
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- | <h2 id="RNA-degradation-experiment" class="protocol-header">RNA | + | <h2 id="RNA-degradation-experiment" class="protocol-header">RNA Degradation Rate</h2> |
<div class="protocol RNA-degradation-experiment"> | <div class="protocol RNA-degradation-experiment"> | ||
The strain producing Spinach was grown in 2 mL LB overnight.</br> | The strain producing Spinach was grown in 2 mL LB overnight.</br> | ||
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- | <h2 id="standard-spinach" class="protocol-header">Spinach | + | <h2 id="standard-spinach" class="protocol-header">Spinach Standard Series</h2> |
<div class="protocol standard-spinach"> | <div class="protocol standard-spinach"> | ||
Spinach produced by <i>in vitro</i> transcription was mixed with DFHBI-1T in different known concentrations. The fluorescence of each sample was measured on a fluorometer. | Spinach produced by <i>in vitro</i> transcription was mixed with DFHBI-1T in different known concentrations. The fluorescence of each sample was measured on a fluorometer. | ||
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- | <h2 id="chemical-transformation-protocol" class="protocol-header">Transformation | + | <h2 id="chemical-transformation-protocol" class="protocol-header">Transformation of Chemically Competent <i>E. coli</i></h2> |
<div class="protocol chemical-transformation-protocol"> | <div class="protocol chemical-transformation-protocol"> | ||
Transformation with chemically competent <i>E. coli</i></br> | Transformation with chemically competent <i>E. coli</i></br> | ||
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<h1>Recipes</h1> | <h1>Recipes</h1> | ||
+ | <h2 id="Agarose-in-TAE" class="protocol-header">1% Agarose in TAE</h2> | ||
+ | <div class="protocol Agarose-in-TAE"> | ||
+ | Mix | ||
+ | <ul> | ||
+ | <li>5 g UltraPure Agarose | ||
+ | <li>50 ml 10X TAE buffer (0.4M Tris, 0.04M Acetate and 0.010M EDTA) | ||
+ | <li>450 ml MilliQ water | ||
+ | </ul> | ||
+ | Heat the mixture in an autoclave or a microwave until the agarose is completely melted. | ||
+ | </div> | ||
+ | <h2 id="Fluorescence-buffer" class="protocol-header">Fluorescence Buffer</h2> | ||
+ | <div class="protocol Fluorescence-buffer"> | ||
+ | Mix the components listed below in MilliQ water to the final concentrations stated, in the final amount needed. | ||
+ | <ul> | ||
+ | <li>20 mM HEPES | ||
+ | <li>100 mM KCl | ||
+ | <li>5 mM NaCl | ||
+ | <li>100 μM MgCl<sub>2</sub> | ||
+ | </ul> | ||
+ | Heat the mixture to 40 °C and shake until complete dissolution is achieved. | ||
+ | </div> | ||
+ | <h2 id="LB" class="protocol-header"> LB Recipies</h2> | ||
+ | <div class="protocol LB"> | ||
+ | LB Lennox Broth<br> | ||
+ | 20 g LB Lennox broth (Sigma Aldrich) were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 °C.<br> | ||
+ | <br> | ||
+ | LB Miller Broth<br> | ||
+ | 20 g LB Lennox broth (Sigma Aldrich) and 5 g NaCl were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 °C. | ||
+ | </div> | ||
- | + | <h2 id="Physiological-saline" class="protocol-header"> Physiological Saline</h2> | |
- | + | <div class="protocol Physiological-saline"> | |
- | + | Mix 9 g NaCl in 1 l MilliQ water and autoclave for 20 minutes at 121 °C. | |
+ | </div> | ||
Latest revision as of 00:53, 18 October 2014
During our laboratory work several protocols and recipes were applied. To make sure that the work is documented in a systematical and reproducible manner, we have made a list of the procedures and added it to this page.
Protocols
Biolector Measurements
The experiment ran for 12.5 hours with measurements of cell density and fluorescence in each well every 5 minutes. The cell density was measured by light scatter at 620 nm and fluorescence was measured at 520 nm with excitation at 488 nm.
DNA Ligation
- 1 μL T4 ligase*
- 2 μL T4 ligase buffer*
- The cut DNA fragments
- Sterile Milli-Q water up to 20 μL
FACS Measurements
The cultures were washed twice in PBS (Phosphate Buffered Saline) and then resuspended in PBS. The suspensions were diluted in PBS to a concentration yielding 1000-1500 events per second.
Fluorometer Measurement
A Shimadzu RF-5301PC Spectrofluorophotometer was used.
For Spinach measurements the instrument was configured with an excitation wavelength of 482 nm and an emission wavelength of 505 nm. The excitation slit was set to 5 and the emission slit was set to 3. The sizes of the slits determine how wide a range of wavelengths is let through the excitation and emission monochromators. Thus a large slit will increase the signal, but will decrease the wavelength precision of the instrument. The fluorometers spectrum function was used to find the largest slits that did not allow significant amounts of excitation light to be detected at the emission wavelength. A cuvette with pure water was used to blank the instrument.
A 10 ml sample of culture was centrifuged at 4000g for 5 min. The pellet was washed twice and resuspended in 1 ml 0.9% NaCl. The OD600 of the sample was then measured and DFHBI-1T was added to a final concentration of 200 μM. After 1 hour on ice, the fluorescence of the sample was measured.
Gel Electrophoresis
1% agarose in 1x TAE buffer is melted and poured into a mold. Ethidium bromide is added to a concentration of 25 μL/L, and is mixed using a pipette tip. (Note: Ethidium bromide is a carcinogen, therefore use gloves when working with the gel. Remove the glove immediately after your work with ethidium bromide in order to avoid contamination of the lab). A comb is placed in the mold to create wells when the agarose solidifies.
When the gel is set, the comb is removed and the gel is transferred to a vessel containing 1x TAE buffer. A suitable DNA ladder is added to the first well, and each sample is then added to an individual well.
When all samples have been loaded, the gel is run with a constant voltage usually 75-100 V for approximately 30 minutes. A lower voltage and a longer duration generally yields better separation.
The gel is analysed with UV light. The gel can then be photographed or bands can be cut out for further work.
Glycerol Stock-Preservation
- Add one mL sterile 50% glycerol to a cryotube
- Add one mL overnight culture to the cryotube
- Store at -80 °C
In vitro Transcription
- Use gloves
- Use pipettes dedicated to RNase-free work
- Use RNase-free microcentrifuge tubes
- Use RNase Zap to clean the bench
- The T7 promoter was added as a tail to the forward primer
- The T7 promoter used was BBa_R0085
- The PCR product was purified with a Qiagen kit and eluted in DEPC-treated water
- 20 μL Transcription Buffer
- 5 μL ATP 10mM
- 5 μL CTP 10mM
- 5 μL GTP 10mM
- 5 μL UTP 10mM
- 150 U T7 RNA polymerase
- 1 μg template DNA
- DEPC-treated water to 100 μL
Microscopy
- Cells are grown O/N in LB media to OD600 of approximately 4
- Cells are washed and resuspended in physiological saline with 200 μM DFHBI-1T
- 1 μl cell suspension is placed on a poly-lysin coated microscope slide and covered with a coverslide
- Cells were visualized with 630x magnification using a GFP filter and UV light
PCR
Plasmid Purification
Preparation of Competent Cells
- 2 mL LB is inoculated with cells from a single colony and incubated at 37 °C O/N at 350 rpm
- 200 mL of preheated (37 °C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 °C and 350 rpm until OD 600 reaches 0.3 - 0.6
- When OD600 reaches 0.3 - 0.6 the shake flask is chilled in an ice-bath until the culture is completely cooled. All steps from here should be kept as cold as possible.
- The 200 mL culture is transferred to 50 mL tubes and spun at 5500 G for 10 minutes. The supernatants are discarded and each pellet is resuspended in 25 mL ice cold 0.1 Molar CaCl2. The tubes are poured together to 2x50 mL.
- The cells are spun at 5500 G for 10 minutes and the supernatant is discarded. The cells are resuspended in 1 mL 0.1 Molar CaCl2 and transferred to microcentrifuge tubes.
- The cells are spun at 15000 G for 2 minutes and the supernatant is discarded. The cells are resuspended in 1 ml 0.1 Molar CaCl2 with 15% glycerol.
- The cells are aliquoted into portions of 200 μL in pre-chilled Eppendorf tubes and immediately stored at -80 °C.
Restriction Digestion
- Each reaction should contain:
- 1X NEBuffer (according to the desired restriction enzyme(s)) with BSA.
- 1 unit/μl restriction enzyme.
- The DNA that is to be cut, final concentration of 10 ng/μL
- Milli-Q water to a volume of 20 μL
- Incubate the reactions at the restriction enzymes’ optimal temperature. If the restriction reaction is analytical, 30 minutes is enough. If the DNA is to be purified, a longer duration is recommended, e.g. 3 hours.
- Add loading buffer to samples and run on 1% agarose gel with ethidium bromide (0.2 μg/ml).
- Analyze your gel, and cut bands out if desired.
RNA Degradation Rate
- Each sample was centrifuged, washed and resuspended in 1 mL 0.9 % NaCl.
- The OD600 of the sample was measured.
- DFHBI-1T was added to a concentration of 200 μM and incubated on ice for 1 hour.
- The fluorescence of the sample was then measured on a fluorometer
Spinach Standard Series
- 20 mM HEPES
- 100 mM KCl
- 5 mM NaCl
- 100 μM MgCl2
Transformation of Chemically Competent E. coli
- Competent cells are defrosted in an ice bath
- 50 μL cells is transferred to a cooled eppendorf tube
- 1-5 μl DNA is transferred to the competent cells, mixed by gently swirling the tube
- DNA and cell mix is kept on ice for 10 minutes
- Cells are heat shocked at 42 °C for 30 seconds in water bath
- Cells are kept on ice for 3 minutes.
- 500 μL LB without antibiotics is added and incubated at 37 °C for 30-120 minutes depending on antibiotic marker
- Plate cells on LB plate with the relevant antibiotic and incubate overnight at 37 °C
Recipes
1% Agarose in TAE
- 5 g UltraPure Agarose
- 50 ml 10X TAE buffer (0.4M Tris, 0.04M Acetate and 0.010M EDTA)
- 450 ml MilliQ water
Fluorescence Buffer
- 20 mM HEPES
- 100 mM KCl
- 5 mM NaCl
- 100 μM MgCl2
LB Recipies
20 g LB Lennox broth (Sigma Aldrich) were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 °C.
LB Miller Broth
20 g LB Lennox broth (Sigma Aldrich) and 5 g NaCl were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 °C.
Physiological Saline