From 2014.igem.org

(Difference between revisions)

Latest revision as of 00:53, 18 October 2014

Protocols & Recipes

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

Cultures of 2 ml LB were inoculated and incubated at 37 °C and 350 RPM O/N.
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).
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

Each reaction of 20 μL contained:

1 μL T4 ligase*
2 μL T4 ligase buffer*
The cut DNA fragments
Sterile Milli-Q water up to 20 μL

The DNA was mixed in a molar ratio of between 3:1 and 5:1 (insert:vector), and the volume of DNA was dependent on the concentration of the fragments and the number of ligations requiring the same fragments. The reactions were incubated at RT for 2-3 hours. To assess the background vector self-ligation rate, we always included controls with no insert added. Furthermore we included controls with no insert, and no ligase to estimate the amount of uncut vector. The ligation products were immediately used to transform cells. * Thermo Scientific

FACS Measurements

2 ml LB media was inoculated with a single colony and inoculated at 37 ° and 350 RPM O/N.
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

To prevent RNase, work in an RNase free environment:

Use gloves
Use pipettes dedicated to RNase-free work
Use RNase-free microcentrifuge tubes
Use RNase Zap to clean the bench

To obtain the DNA template for the transcription we used PCR amplification

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

Each transcription reaction mix consisted of:

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

The reactions were incubated at 37°C for 2-3 hours, and stopped with the addition of 3 μL EDTA. The product was purified with Qiagen RNeasy Kit, and eluted in 50 μL DEPC-treated water.

Microscopy

Microscopy was used to visualize fluorescent cells, both with GFP and Spinach.

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

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 here.

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.
Good aeration is important for obtaining high plasmid concentrations, so tube inclination should optimally be 45 °.
The purification was done with a Zymo Zyppy Plasmid Miniprep Kit, by following the manufacturer’s manual.

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

The strain producing Spinach was grown in 2 mL LB overnight.
This culture was used to inoculate 200 mL LB in a shake flask which was incubated at 37 °C for 4 hours.
Rifampicin was then added to a final concentration of 100 μg/mL, at t = 0. Samples of 10 mL were taken every hour:

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

Background fluorescence level from cells was established by measuring a similarly treated sample from a strain that did not produce spinach.

Spinach Standard Series

Spinach produced by in vitro transcription was mixed with DFHBI-1T in different known concentrations. The fluorescence of each sample was measured on a fluorometer. A buffer was added to a final concentration of:

20 mM HEPES
100 mM KCl
5 mM NaCl
100 μM MgCl₂

The samples were kept on ice to minimize temperature-dependent fluorescence variations.

Transformation of Chemically Competent E. coli

Transformation with chemically competent E. coli

IMPORTANT: Keep everything on ice unless otherwise stated in the protocol.

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

Mix

5 g UltraPure Agarose
50 ml 10X TAE buffer (0.4M Tris, 0.04M Acetate and 0.010M EDTA)
450 ml MilliQ water

Heat the mixture in an autoclave or a microwave until the agarose is completely melted.

Fluorescence Buffer

Mix the components listed below in MilliQ water to the final concentrations stated, in the final amount needed.

20 mM HEPES
100 mM KCl
5 mM NaCl
100 μM MgCl₂

Heat the mixture to 40 °C and shake until complete dissolution is achieved.

LB Recipies

LB Lennox Broth
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

Mix 9 g NaCl in 1 l MilliQ water and autoclave for 20 minutes at 121 °C.

Number of Reactions:
MQ water	μL
5X HF buffer	μL
2mM dNTP	μL
Polymerase	μL
Template	μL
10μM Primer x2	μL

@@ Line 2: / Line 2: @@
 <html>
+<script>
+$(document).ready(function() {
+    $("#PCR-input").trigger("input");
+    $(window).keydown(function(event){
+        if(event.keyCode == 13) {
+            event.preventDefault();
+            return false;
+        }
+    });
+});
+</script>
+<head>
+</head>
 <body>
 <div class="pagescontent">
@@ Line 7: / Line 27: @@
 <pageheader>Protocols & Recipes</pageheader>
 <maincontent>
-<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.
+<br><br>
+<h1>Protocols</h1>
-<h1>Protocols</h1>
+<h2 id="BioLector" class="protocol-header">Biolector Measurements</h2>
-<h2 id="glycerol-stock-protocol" class="protocol-header">Glycerol stock-preservation</h2>
+<div class="protocol BioLector">
-<div class="protocol glycerol-stock-protocol">
+Cultures of 2 ml LB were inoculated and incubated at 37 &deg;C and 350 RPM O/N.</br>
-<ul>
+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 &deg;C and 1000 RPM ensuring optimal mass transfer (oxygen).<br>
-    <li>Add one mL sterile 50% glycerol to a cryotube</li>
+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.
-    <li>Add one mL overnight culture to the cryotube</li>
-    <li>Store at -80 °C</li>
-</ul>
 </div>
-<h2 id="standard-spinach" class="protocol-header">Spinach standard series</h2>
-<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.
-A buffer was added to a final concentration of:
+<h2 id="Protocol-for-ligation-for-DNA-cloning" class="protocol-header">DNA Ligation</h2>
+<div class="protocol Protocol-for-ligation-for-DNA-cloning">
+Each reaction of 20 &mu;L contained:
 <ul>
-     <li>20 mM HEPES</li>
+     <li>1 &mu;L T4 ligase*</li>
-     <li>100 mM KCl</li>
+     <li>2 &mu;L T4 ligase buffer*</li>
-     <li>5 mM NaCl</li>
+     <li>The cut DNA fragments</li>
-     <li>100 &mu;M MgCl<sub>2</sub></li>
+     <li>Sterile Milli-Q water up to 20 &mu;L</li>
 </ul>
-The samples were kept on ice to minimize temperature-dependent fluorescence variations.
+The DNA was mixed in a molar ratio of between 3:1 and 5:1 (insert:vector), and the volume of DNA was dependent on the concentration of the fragments and the number of ligations requiring the same fragments.
+The reactions were incubated at RT for 2-3 hours.
+To assess the background vector self-ligation rate, we always included controls with no insert added. Furthermore we included controls with no insert, and no ligase to estimate the amount of uncut vector.
+The ligation products were immediately used to transform cells.
+* Thermo Scientific
 </div>
+<h2 id="FACS" class="protocol-header">FACS Measurements</h2>
+<div class="protocol FACS">
+ml LB media was inoculated with a single colony and inoculated at 37 &deg; 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 measurement</h2>
+<h2 id="fluorometer-measurement-protocol" class="protocol-header">Fluorometer Measurement</h2>
 <div class="protocol fluorometer-measurement-protocol">
 <p>
@@ Line 49: / Line 84: @@
 </div>
-<h2 id="RNA-degradation-experiment" class="protocol-header">RNA degradation experiment</h2>
-<div class="protocol RNA-degradation-experiment">
-The strain producing Spinach was grown in 2 mL LB overnight.</br>
+<h2 id="gel-protocol" class="protocol-header">Gel Electrophoresis </h2>
-This culture was used to inoculate 200 mL LB in a shake flask which was incubated at 37 &deg;C for 4 hours.</br>
+<div class="gel-protocol protocol">
-Rifampicin was then added to a final concentration of 100 &mu;g/mL, at t = 0.
+<p>
-Samples of 10 mL were taken every hour:
+% agarose in 1x TAE buffer is melted and poured into a mold. Ethidium bromide is added to a concentration of 25 &mu;L/L, and is mixed using a pipette tip. (<b>Note: Ethidium bromide is a carcinogen, therefore use gloves when working with the gel.</b> Remove the glove immediately after your work with ethidium bromide in order to avoid contamination of the lab).</br>
+A comb is placed in the mold to create wells when the agarose solidifies.
+</p>
+<p>
+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.
+</p>
+<p>
+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.
+</p>
+<p>
+The gel is analysed with UV light. The gel can then be photographed or bands can be cut out for further work.
+</p>
+</div>
+<h2 id="glycerol-stock-protocol" class="protocol-header">Glycerol Stock-Preservation</h2>
+<div class="protocol glycerol-stock-protocol">
 <ul>
-     <li>Each sample was centrifuged, washed and resuspended in 1 mL 0.9 % NaCl.</li>
+     <li>Add one mL sterile 50% glycerol to a cryotube</li>
-     <li>The OD600 of the sample was measured.</li>
+     <li>Add one mL overnight culture to the cryotube</li>
-     <li>DFHBI-1T was added to a concentration of 200 &mu;M and incubated on ice for 1 hour.</li>
+     <li>Store at -80 °C</li>
-    <li>The fluorescence of the sample was then measured on a fluorometer</li>
 </ul>
-Background fluorescence level from cells was established by measuring a similarly treated sample from a strain that did not produce spinach.
 </div>
-<h2 id="Protocol-for-ligation-for-DNA-cloning" class="protocol-header">Protocol for ligation for DNA cloning</h2>
-<div class="protocol Protocol-for-ligation-for-DNA-cloning">
-Each reaction of 20 &mu;L contained:
+<h2 id="IVT-protocol" class="protocol-header"><i>In vitro</i> Transcription</h2>
+<div class="protocol IVT-protocol">
+To prevent RNase, work in an RNase free environment:
 <ul>
-     <li>1 &mu;L T4 ligase*</li>
+     <li>Use gloves</li>
-     <li>2 &mu;L T4 ligase buffer*</li>
+     <li>Use pipettes dedicated to RNase-free work</li>
-     <li>The cut DNA fragments</li>
+     <li>Use RNase-free microcentrifuge tubes</li>
-     <li>Sterile Milli-Q water up to 20 &mu;L</li>
+     <li>Use RNase Zap to clean the bench</li>
 </ul>
-The DNA was mixed in a molar ratio of between 3:1 and 5:1 (insert:vector), and the volume of DNA was dependent on the concentration of the fragments and the number of ligations requiring the same fragments.
-The reactions were incubated at RT for 2-3 hours.
-To assess the background vector self-ligation rate, we always included controls with no insert added. Furthermore we included controls with no insert, and no ligase to estimate the amount of uncut vector.
-The ligation products were immediately used to transform cells.
+To obtain the DNA template for the transcription we used PCR amplification
+<ul>
+    <li>The T7 promoter was added as a tail to the forward primer</li>
+    <li>The T7 promoter used was BBa_R0085</li>
+    <li>The PCR product was purified with a Qiagen kit and eluted in DEPC-treated water</li>
+</ul>
-* Thermo Scientific
+Each transcription reaction mix consisted of:
+<ul>
+    <li>20 &mu;L Transcription Buffer</li>
+    <li>5 &mu;L ATP 10mM</li>
+    <li>5 &mu;L CTP 10mM</li>
+    <li>5 &mu;L GTP 10mM</li>
+    <li>5 &mu;L UTP 10mM</li>
+    <li>150 U T7 RNA polymerase</li>
+    <li>1 &mu;g template DNA</li>
+    <li>DEPC-treated water to 100 &mu;L</li>
+</ul>
+The reactions were incubated at 37&deg;C for 2-3 hours, and stopped with the addition of 3 &mu;L EDTA.
+The product was purified with Qiagen RNeasy Kit, and eluted in 50 &mu;L DEPC-treated water.
 </div>
-<h2 id="Plasmid-purification" class="protocol-header">Plasmid purification</h2>
-<div class="protocol Plasmid-purification">
+<h2 id="microscopy-protocol" class="protocol-header">Microscopy</h2>
-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 &deg;C overnight.</br>
+<div class="protocol microscopy-protocol">
-Good aeration is important for obtaining high plasmid concentrations, so tube inclination should optimally be 45 &deg;.</br>
+Microscopy was used to visualize fluorescent cells, both with GFP and Spinach.
-The purification was done with a Zymo Zyppy Plasmid Miniprep Kit, by following the manufacturer’s manual.
+<br>
+<ul>
+    <li>Cells are grown O/N in LB media to OD600 of approximately 4</li>
+    <li>Cells are washed and resuspended in physiological saline with 200 &mu;M DFHBI-1T</li>
+    <li>1 &mu;l 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>
+</ul>
 </div>
 <h2 id="PCR-protocol" class="protocol-header">PCR</h2>
 <div class="protocol PCR-protocol">
-<center><img src="https://static.igem.org/mediawiki/2014/8/8e/PCR_PIC.png" width="942" height="180"/></center></br>
+<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">
-<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">
-A master mix is made with final concentration of:</br>
+A master mix was made with final concentration of:</br>
 X HF buffer</br>
 .2mM dNTP</br>
@@ Line 102: / Line 174: @@
 .02 units/ml polymerase</br>
 </br>
-The concentration of template is allowed to vary and can almost never be to low. Use our calculator below to calculate the amount of solutes to be added to your mastermix. Remember its always a good idea to make mastermix for an additional reaction than need.
+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>
 <table width="350px" border="0" cellspacing="5" cellpadding="0" style="background-color: transparent;">
    <tr colspan="2">
@@ Line 146: / Line 218: @@
 </form>
-<ul>
-<li>Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.</li>
+</br>
-<li>The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.</li>
+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 &deg;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>.
-<li>Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.</li>
-<li>Mark the PCR tubes on lid and side and place in thermocycler.</li>
-<li>The annealing temperature should match the primer melting temperatures minus 5 degrees. This can be hard since Tm predictors tend to disagree.</li>
-<li>Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).</li>
-</ul>
 </div>
-<h2 id="gel-protocol" class="protocol-header">Gel electrophoresis </h2>
-<div class="gel-protocol protocol">
-<p>
+<h2 id="Plasmid-purification" class="protocol-header">Plasmid Purification</h2>
-% agarose in 1x TAE buffer is melted and poured into a mold. Ethidium bromide is added to a concentration of 25 &mu;L/L, and is mixed using a pipette tip. (<b>Note: Ethidium bromide is a carcinogen, therefore use gloves when working with the gel.</b> Remove the glove immediately after your work with ethidium bromide in order to avoid contamination of the lab).</br>
+<div class="protocol Plasmid-purification">
-A comb is placed in the mold to create wells when the agarose solidifies.
+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 &deg;C overnight.</br>
-</p>
+Good aeration is important for obtaining high plasmid concentrations, so tube inclination should optimally be 45 &deg;.</br>
-<p>
+The purification was done with a Zymo Zyppy Plasmid Miniprep Kit, by following the manufacturer’s manual.
-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.
-</p>
-<p>
-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.
-</p>
-<p>
-The gel is analysed with UV light. The gel can then be photographed or bands can be cut out for further work.
-</p>
 </div>
-<h2 id="m9-protocol" class="protocol-header">Recipe on M9 minimal media</h2>
-<div class="protocol m9-protocol">
+<h2 id="competent-cells-protocol" class="protocol-header">Preparation of Competent Cells</h2>
-<ol>
+<div class="protocol competent-cells-protocol">
-<li>Make M9 salts</li>
+<ul>
-<li>To make M9 Salts aliquot 800ml H2O and add</li>
+    <li>2 mL LB is inoculated with cells from a single colony and incubated at 37 &deg;C O/N at 350 rpm</li>
-        <ul>
+    <li>200 mL of preheated (37 &deg;C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 &deg;C and 350 rpm until OD 600 reaches 0.3 - 0.6</li>
-            <li>64g Na2HPO4-7H2O</li>
+    <li>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.</li>
-            <li>15g KH2PO4</li>
+    <li>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 CaCl<sub>2</sub>. The tubes are poured together to 2x50 mL.</li>
-            <li>2.5g NaCl</li>
+    <li>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 CaCl<sub>2</sub> and transferred to microcentrifuge tubes.</li>
-            <li>5.0g NH4Cl</li>
+    <li>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.</li>
-            <li>Stir until dissolved</li>
+    <li>The cells are aliquoted into portions of 200 &mu;L in pre-chilled Eppendorf tubes and immediately stored at -80 &deg;C.</li>
-            <li>Adjust to 1000ml with distilled H2O</li>
+</ul>
-            <li>Sterilize by autoclaving</li>
-                       </ul>
-<li>Measure ~700ml of distilled H2O (sterile)</li>
-<li>Add 200ml of M9 salts</li>
-<li>Add 2ml of 1M MgSO4 (sterile)</li>
-<li>Add 20 ml of 20% glucose (or other carbon source)</li>
-<li>Add 100ul of 1M CaCl2 (sterile)</li>
-<li>Adjust to 1000ml with distilled H2O</li>
-</ol>
 </div>
-<h2 id="restriction-protocol" class="protocol-header">Restriction digestion</h2>
+<h2 id="restriction-protocol" class="protocol-header">Restriction Digestion</h2>
 <div class="protocol restriction-protocol">
 <ol>
@@ Line 213: / Line 264: @@
 </div>
-<h2 id="competent-cells-protocol" class="protocol-header">Preparation of competent cells</h2>
-<div class="protocol competent-cells-protocol">
+<h2 id="RNA-degradation-experiment" class="protocol-header">RNA Degradation Rate</h2>
+<div class="protocol RNA-degradation-experiment">
+The strain producing Spinach was grown in 2 mL LB overnight.</br>
+This culture was used to inoculate 200 mL LB in a shake flask which was incubated at 37 &deg;C for 4 hours.</br>
+Rifampicin was then added to a final concentration of 100 &mu;g/mL, at t = 0.
+Samples of 10 mL were taken every hour:
 <ul>
-     <li>2 mL LB is inoculated with cells from a single colony and incubated at 37 &deg;C O/N at 350 rpm</li>
+     <li>Each sample was centrifuged, washed and resuspended in 1 mL 0.9 % NaCl.</li>
-    <li>200 mL of preheated (37 &deg;C) LB is inoculated with 10 ul culture from step 1 in a shake flask and incubated at 37 &deg;C and 350 rpm until OD 600 reaches 0.3 - 0.6</li>
+     <li>The OD600 of the sample was measured.</li>
-     <li>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.</li>
+     <li>DFHBI-1T was added to a concentration of 200 &mu;M and incubated on ice for 1 hour.</li>
-     <li>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 CaCl<sub>2</sub>. The tubes are poured together to 2x50 mL.</li>
+     <li>The fluorescence of the sample was then measured on a fluorometer</li>
-    <li>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 CaCl<sub>2</sub> and transferred to microcentrifuge tubes.</li>
-     <li>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.</li>
-    <li>The cells are aliquoted into portions of 200 &mu;L in pre-chilled Eppendorf tubes and immediately stored at -80 &deg;C.</li>
 </ul>
+Background fluorescence level from cells was established by measuring a similarly treated sample from a strain that did not produce spinach.
 </div>
-<h2 id="chemical-transformation-protocol" class="protocol-header">Transformation with chemically competent <i>E. coli</i></h2>
+<h2 id="standard-spinach" class="protocol-header">Spinach Standard Series</h2>
+<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.
+A buffer was added to a final concentration of:
+<ul>
+    <li>20 mM HEPES</li>
+    <li>100 mM KCl</li>
+    <li>5 mM NaCl</li>
+    <li>100 &mu;M MgCl<sub>2</sub></li>
+</ul>
+The samples were kept on ice to minimize temperature-dependent fluorescence variations.
+</div>
+<h2 id="chemical-transformation-protocol" class="protocol-header">Transformation of Chemically Competent <i>E. coli</i></h2>
 <div class="protocol chemical-transformation-protocol">
 Transformation with chemically competent <i>E. coli</i></br>
@@ Line 244: / Line 313: @@
 </div>
-<h2 id="IVT-protocol" class="protocol-header"><i>In vitro</i> transcription</h2>
-<div class="protocol IVT-protocol">
-To prevent RNase, work in an RNase free environment:
-<ul>
-    <li>Use gloves</li>
-    <li>Use pipettes dedicated to RNase-free work</li>
-    <li>Use RNase-free microcentrifuge tubes</li>
-    <li>Use RNase Zap to clean the bench</li>
-</ul>
-To obtain the DNA template for the transcription we used PCR amplification
-<ul>
-    <li>The T7 promoter was added as a tail to the forward primer</li>
-    <li>The T7 promoter used was BBa_R0085</li>
-    <li>The PCR product was purified with a Qiagen kit and eluted in DEPC-treated water</li>
-</ul>
-Each transcription reaction mix consisted of:
+<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>20 &mu;L Transcription Buffer</li>
+<li>5 g UltraPure Agarose
-    <li>5 &mu;L ATP 10mM</li>
+<li>50 ml 10X TAE buffer (0.4M Tris, 0.04M Acetate and 0.010M EDTA)
-    <li>5 &mu;L CTP 10mM</li>
+<li>450 ml MilliQ water
-    <li>5 &mu;L GTP 10mM</li>
-    <li>5 &mu;L UTP 10mM</li>
-    <li>150 U T7 RNA polymerase</li>
-    <li>1 &mu;g template DNA</li>
-    <li>DEPC-treated water to 100 &mu;L</li>
 </ul>
+Heat the mixture in an autoclave or a microwave until the agarose is completely melted.
-The reactions were incubated at 37&deg;C for 2-3 hours, and stopped with the addition of 3 &mu;L EDTA.
-The product was purified with Qiagen RNeasy Kit, and eluted in 50 &mu;L DEPC-treated water.
 </div>
-<h2 id="microscopy-protocol" class="protocol-header">Microscopy</h2>
-<div class="protocol microscopy-protocol">
+<h2 id="Fluorescence-buffer" class="protocol-header">Fluorescence Buffer</h2>
-Microscopy was used to visualize fluorescent cells, both with GFP and Spinach.
+<div class="protocol Fluorescence-buffer">
-<br>
+Mix the components listed below in MilliQ water to the final concentrations stated, in the final amount needed.
 <ul>
-    <li>Cells are grown O/N in LB media to OD600 of approximately 4</li>
+<li>20 mM HEPES
-    <li>Cells are washed in PBS and resuspended in M9 media with 200 &mu;M DFHBI-1T</li>
+<li>100 mM KCl
-    <li>1 &mu;l cell suspension is placed on a poly-lysin coated microscope slide and covered with a coverslide</li>
+<li>5 mM NaCl
-    <li>Cells were visualized with 630x magnification using a GFP filter and UV light</li>
+<li>100 &mu;M MgCl<sub>2</sub>
 </ul>
+Heat the mixture to 40 &deg;C and shake until complete dissolution is achieved.
 </div>
-<h1>Recipes</h1>
-<h2 id="test2" class="protocol-header">test3</h2>
-<div class="LB protocol">
-Microscopy was used to visualize fluorescent cells, both with GFP and Spinach.
+<h2 id="LB" class="protocol-header"> LB Recipies</h2>
+<div class="protocol LB">
+LB Lennox Broth<br>
+g LB Lennox broth (Sigma Aldrich) were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 &deg;C.<br>
 <br>
-<ul>
+LB Miller Broth<br>
-    <li>Cells are grown O/N in LB media to OD600 of approximately 4</li>
+g LB Lennox broth (Sigma Aldrich) and 5 g NaCl were mixed with 1 l MilliQ water and autoclaved for 20 minutes at 121 &deg;C.
-    <li>Cells are washed in PBS and resuspended in M9 media with 200 &mu;M DFHBI-1T</li>
+</div>
-    <li>1 &mu;l 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>
-</ul>
+<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 &deg;C.
 </div>

Team:DTU-Denmark/Methods/Protocols