Difference between revisions of "Team:Kent/Experiments"

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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
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<div id="navbox">
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 +
            <ul>
 +
                <li>
 +
                    <a href="#">Project</a>
 +
                    <ul class="drop-menu menu-1">
 +
                        <a href="https://2017.igem.org/Team:Kent/Description"><li>Description</li></a>
 +
<a href="https://2017.igem.org/Team:Kent/Model"><li>Modelling</li></a>
 +
                      <a href="https://2017.igem.org/Team:Kent/Results"><li>Results</li></a>
 +
                       
 +
 +
                    </ul>
 +
                <li>
 +
                    <a href="#">Parts</a>
 +
                    <ul class="drop-menu menu-2">
 +
 +
                        <a href="https://2017.igem.org/Team:Kent/Basic_Part"><li>Basic Parts</li></a>
 +
                       
 +
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Notebook</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Notebook"><li>Logbook</li></a>
 +
<a href="https://2017.igem.org/Team:Kent/Experiments"><li>Experiments</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Improve"><li>Future Ideas</li></a>
 +
                       
 +
                    </ul>
 +
                </li>
 +
                <li id="teamLogo">
 +
                    <a href="https://2017.igem.org/Team:Kent">
 +
                        <h1><img src="https://static.igem.org/mediawiki/2017/thumb/4/4f/T--Kent--Kenttemplogo.png/667px-T--Kent--Kenttemplogo.png"></h1>
 +
                    </a>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Safety</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Safety"><li>Project Safety</li></a>
 +
                       
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Team</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/Team"><li>Meet the Team</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Contribution"><li>Contribution</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Attributions"><li>Attribution</li></a>
 +
                    </ul>
 +
                </li>
 +
                <li>
 +
                    <a href="#">Human Practices</a>
 +
                    <ul class="drop-menu menu-2">
 +
                        <a href="https://2017.igem.org/Team:Kent/HP/Silver"><li>Silver</li></a>
 +
<a href="https://2017.igem.org/Team:Kent/HP/Gold_Integrated"><li>Gold</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Engagement"><li>Public Engagement</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/InterLab"><li>Interlab</li></a>
 +
                        <a href="https://2017.igem.org/Team:Kent/Collaborations"><li>Collaboration</li></a>
 +
                    </ul>
 +
                </li>
 +
            </ul>
 +
        </nav>
 
</div>
 
</div>
 +
        <div id ="title">
 +
                <img src = "https://static.igem.org/mediawiki/2017/0/06/T--Kent--protocol_icon.png" id="img1">
 +
<span>Experiments & Protocols </span>
 +
<img src = "https://static.igem.org/mediawiki/2017/3/3e/T--Kent--protocol_icon2.png" id="img2">
 +
        </div>
 +
        <nav class="droptext arrows">
 +
<header class="hull">
 +
<label for="acc-close" class="hull-title">Basic Protocols</label>
 +
</header>
 +
<input type="radio" name="droptext" id="cb1" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb1">Production of Lysogeny broth (LB)</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">For 1 litre of LB a mixture of 10g of sodium chloride, 10g peptone, 5g of yeast extract as well as 1
 +
litre of distilled water in a glass bottle. We then used a magnetic spinner to help mix the powders
 +
with the water, we avoided shaking the glass bottle as it would cause froth and waste some of the
 +
LB.
 +
<br>
 +
When making the LB we also made another litre batch and added 15g of agar extract to be able to
 +
grow bacteria on plates.</div>
 +
</section>
 +
<input type="radio" name="droptext" id="cb2" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb2">Production of SOB medium and magnesium stock</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">Bringing together 20g of tryptone, 5g of yeast extract, 0.584g of NaCl, 0.186g of KCl and mixing it
 +
with 990ml of millipure water (using the magnetic mixer again) which was then put in to autoclave
 +
to sterilise it, after it was taken out and let for it to cool down to below 60 o C.
 +
<br>
 +
10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm
 +
filter sterilize was then used</div>
 +
</section>
 +
<input type="radio" name="droptext" id="cb3" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb3">Production of SOC medium and glucose stock</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">Once again bring 20g of tryptone, 5g of yeast of extract, 0.584g of NaCl, 0.186g of KCL, and then
 +
bring 970 ml with millipure water and use the magnetic mixer once again, this was also then put in
 +
to autoclave.
 +
<br>
 +
10ml of 2M Mg 2+ stock and then bring it to 100ml with milllipure water, filter sterilize it with 0.2m
 +
and then final add 20ml of 1M glucose stock.</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb4" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb4">Production of Glycerol stock</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">If you wish to store bacteria long term, you will need to create a Glycerol Stock after
 +
inoculating an overnight liquid culture
 +
<br>
 +
<ul><li>Once bacterial growth has been achieved, 500μL of the overnight liquid
 +
culture needs to be added to 500μL of 50% glycerol in a 2mL tube where it
 +
should be gently mixed</li>
 +
<li>The glycerol stock should then be frozen at -80 o C<ul>
 +
<li> Successive freeze and thaw cycles will reduce the stocks shelf life</li></ul>
 +
</li></ul></div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb5" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb5">Running Agarose Gel</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.
 +
<br>
 +
<ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
 +
250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
 +
seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
 +
much. Make up the evaporated volume to 50ml with distilled water.</li>
 +
<li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
 +
<li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
 +
a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
 +
<li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
 +
solidify (maximum 30 mins)</li>
 +
<li>When the gel has set, remove the comb from the tank (gently!) and then cover the whole
 +
tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
 +
<li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
 +
tube of this and load the whole tube) into well 1( left hand side) and then choose what you
 +
load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
 +
<li>Load all of your digests into the wells 2,3, and 4.</li>
 +
<li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
 +
don’t matter.</li>
 +
<li>Once the visible markers have reached the half way point of the tank, turn off the power
 +
supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
 +
UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb6" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb6">Overnights protocol</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">After a transformation has been run and plates have been streaked and patched, overnight cultures will need to be made:
 +
<br>
 +
<ul><li>Add 10mL of LB broth (not agar) into as many autoclaved conical flasks as
 +
needed</li>
 +
<li>Add 10uL of Chloramphenicol into each conical flask as well</li>
 +
<li>Using a pipette tip, scrape up some of the cell colonies on the agar plates
 +
prepared beforehand and drop it into the conical flask</li>
 +
<li>Cover up the flask using aluminum foil</li>
 +
<li>Incubate the cultures at 37oC and 180 rpm</li>
 +
</ul></div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb7" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb7">Protocol for transformation/ heat shock</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
This requires chemically competent cells to be made beforehand. These must be stored at -80
 +
degrees Celsius:
 +
<br>
 +
<ul><li>First, the competent cells and the plasmid intended for transformation must be thawed on
 +
ice. Additionally, some 1.5ml Eppendorf tubes should be chilled on ice, along with some
 +
pipette tips.</li>
 +
<li>100ul of the chemically competent cells are then pipetted (using the chilled pipette tip) into
 +
a chilled Eppendorf tube.</li>
 +
<li>5ul of DNA is the pipetted into the tube with a chilled tip. This tube is then stored on ice for
 +
30 minutes.</li>
 +
<li>The tube is then placed in a water bath at 42 degrees Celsius for precisely 90 seconds. After
 +
90 seconds is up, the tube is transferred back to ice for 2 minutes.</li>
 +
<li>900ul SOC medium is then added into the tube (with a normal pipette tip, doesn’t need to
 +
be chilled) and then mix very gently by pipetting up and down inside the tube.</li>
 +
<li>The tube is then incubated at 37 degrees Celsius for 45 minutes. The tube should not be
 +
shaken at all at this point.</li>
 +
<li>100ul of the transformation mix is then pipetted into the centre of a plate containing LB agar
 +
and the appropriate antibiotic (for example, for plasmid pSB1C3, Chlorophenicol should be
 +
used, and for pSB1A3, use Ampicillin). Use 1ul of antibiotic for each ml of agar.</li>
 +
<li>In sterile conditions (Bunsen burner, gloves cleaned with IMS (ALLOWED TO DRY)), spread
 +
the bacteria around the plate by keeping the lid as closed as possible and inserting the
 +
spreader then turning the plate around to spread the cells. Then immediately close and
 +
store upside down.</li>
 +
<li>The remained of the transformation mix is then spun at the highest possible speed for 2
 +
minutes. The resulting pellet is then resuspended in 100ul of the existing medium and plated
 +
onto the LB and antibiotic plate.</li>
 +
<li>Incubate in a 37-degree Celsius incubator for 16-18 hours.</li>
 +
<li>Any colonies that result from this should be plated on a patch plate.</li>
 +
<li>This is done by taking a plate of LB agar with the appropriate antibiotic and dividing it up
 +
into sections by drawing a grid on the bottom. These sections are numbered and then using
 +
a sterile pipette tip, the colonies are gently streaked in each section- 1 colony per section.</li></ul></div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb8" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb8">Transformation Guidelines (QuickChange Protocol)</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
Store all XL1-Blue supercompetent cells at -80 o C (prevents loss of efficiency) as they are
 +
sensitive to the smallest of temperature variations, even transferring tubes from one freezer
 +
to another will result in loss of efficiency
 +
<br>
 +
Storage Conditions:
 +
<ul><li>XL1-Blue supercompetent cells should be stored at the bottom of a -80 o C freezer</li>
 +
<li>They should be placed at -80 o C directly from dry ice shipping container</li>
 +
<br>
 +
Aliquoting Cells
 +
<li>Keep the XL1-Blue supercompetent cells on ice at all times</li>
 +
<li>Essential that BD-Falcon polypropylene tubes are places on ice before cells are
 +
thawed</li>
 +
<li>Cells must be aliquoted directly into prechilled tubes (Use of 14-mL BD Falcon Polypropylene Round-Bottom Tubes)</li>
 +
<li>These tubes must be used (BD Biosciences Catalog #352059) for the transformation
 +
protocol</li>
 +
<li>The heat-pulse steps’ duration is critical and has been optimized for the thickness as
 +
well as shape of these tubes</li>
 +
<br>
 +
Length of Heat Pulse
 +
<li>Optimal efficiencies observed when cells are heat pulsed for 45 seconds</li>
 +
<li>Heat pulsing for at least 45 seconds is recommended, allowing for slight variations
 +
in incubation length</li>
 +
<li>Efficiencies noted to decrease sharply when pulsed for &lt;30 seconds or &gt;45 seconds</li>
 +
<li>This defined window of highest efficiency for the XL1-Blue cells results from heat
 +
pulse in step 3 of transformation protocol</li>
 +
<br>
 +
Preparing Agar Plates for Colour Screening
 +
<li>To the LB agar, add <ul><li>80 µg/ml of 5-bromo- 4-chloro- 3-indolyl- β-D-galactopyranoside (X-gal)</li>
 +
<li>20 mM isopropy-1- thio β-D galactopyranoside (IPTG)</li>
 +
<li>Appropriate antibiotic</li></ul></li>
 +
<li>These are all added to prepare the LB agar plates for blue-white colour screening </li>
 +
<li>Alternatively <ul><li>100 μl of 10 mM IPTG and 100 μl of 2% X-gal can be spread on LB agar
 +
plates 30 minutes prior to plating transformations</li></ul> </li>
  
−
<div class="column half_size">
+
<li>The IPTG must be prepared in sterile dH2O </li>
−
<h5>What should this page contain?</h5>
+
<li>The X-gal must be prepared in dimethylformamide (DMF) </li>
 +
<li>IPTG and X-gal MUST NOT be mixed before being pipetted onto the plates since the
 +
chemicals may precipitate.</li>
 +
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
</nav>
 +
<div class="connector">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/b/bf/T--Kent--ExperimentsConnect.png/133px-T--Kent--ExperimentsConnect.png">
 +
</div>
 +
<nav class="droptext arrows">
 +
<header class="hull">
 +
<label for="acc-close" class="hull-title">Interlab Protocols</label>
 +
</header>
 +
<input type="radio" name="droptext" id="cb10" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb10">Calibration of OD 600 Reference Point</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
LUDOX-S40 must be used as a single point reference with the aim to attain a
 +
ratiometric conversion factor, which in turn will be used to transform absorbance
 +
data into standard OD 600 measurement.
 +
<br>
 +
Standard 1 cm path length spectrophotometer readings are instrument dependent,
 +
while plate readers possess a path length less than 1 cm and are volume dependent.
 +
<br>
 +
Therefore, in this situation, there are 2 key objectives:
 +
<ul><li>Ratiometric conversion to transform Abs 600 measurements into OD 600
 +
measurements</li>
 +
<li> Accounting for instrument differences</li></ul>
 +
<br>
 +
Before starting the protocol, path length correction must be switched off. This is
 +
because scattering increases with longer wavelengths therefore adjustment is
 +
confounded by scattering solutions such as dense cells. However, many plate
 +
readers have automatic path length correction which is based on volume
 +
adjustment that uses ratio of absorbance measurements at 900 + 950 nm.
 +
LUDOX solution is only weakly scattering so will produce low absorbance values
 +
<br>
 +
Use same cuvettes, plates and volumes that are going to be used in cell based assays
 +
<br>
 +
Materials:
 +
<ul><li>1 mL 100% LUDOX</li>
 +
<li>H 2 O</li>
 +
<li>96 well plate (black with flat, transparent/clear bottom)</li></ul>
 +
<br>
 +
Method
 +
<ul><li>100 µl of LUDOX should be added into wells A1, B1, C1 and D2 (or 1mL into
 +
cuvette)</li>
 +
<li>100 µl of H 2 O should be added into wells A2, B2, C2 and D2</li>
 +
<li>Measure absorbance 600nm of all samples in all standard measurement modes in instrument</li>
 +
<li>Record data</li>
 +
</div>
 +
</section>
 +
 +
<input type="radio" name="droptext" id="cb11" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb11">Production of Fluorescein stock solution</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 
<ul>
 
<ul>
−
<li> Protocols </li>
+
<li> Spin down the Fluorescein stock tube and ensure the pellet is at the tubes' bottom </li>
−
<li> Experiments </li>
+
<li>Prepare 2x fluorescein stock solution (100 µM)<ul>
−
<li> Documentation of the development of your project </li>
+
<li>Resuspend Fluorescein in 1mL 1xPBS</li>
 +
<li>Ensure Fluorescein is properly dissolved<br>
 +
After the resuspension, pipette up and down and examine the solution in
 +
the tip (if particulates are visisble, continue to mix solution until they
 +
disappear)</li></ul></li>
 +
<br>
 +
<li>Dilute the 2x Fluorescein stock solution<ul>
 +
<li> With 1xPBS to make 1x fluorescein solution</li>
 +
<li>With resulting concentration of fluorescein stock solution 50 µM
 +
(500 µL of 2x fluorescein in 500 µL 1x PBS to make 1 mL of 50 µM (1x)
 +
fluorescein solution)</li></ul></li>
 
</ul>
 
</ul>
−
 
 
</div>
 
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb12" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb12">Fluorescein Fluorescence Standard Curve</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
A dilution series of Fluorescein in 4 replicates must be prepared where the
 +
fluorescence is measured in a 96 well plate in standard mode on a plate reader. A
 +
standard curve will be generated of fluorescence of fluorescein concentration. This
 +
will be used to correct cell based readings to an equivalent fluorescein
 +
concentration, which will then be converted into a GFP concentration.
 +
<br>
 +
<br>
 +
Materials
 +
<br>
 +
<ul><li>Fluorescein</li>
 +
<li>10mL 1xPBS (Phosphate Buffered Saline)</li>
 +
<li>96 well plate (black with flat, transparent/clear bottom)</li></ul>
 +
<br>
 +
Method
 +
<br>Serial dilutions need to be performed across columns 1-11
 +
Column 12 must contain PBS buffer only
 +
<br>
 +
The plate will initially be setup fluorescein stock in column 1 and equal volume of1xPBS in columns 2-12
 +
<ul><li> Add 100 µL of PBS into wells A2-A12, B2-B12, C2-C12 and D2-D12</li>
 +
<li>Add 200 µL of Fluorescein 1x stock solution into A1, B1, C1 and D1</li>
  
−
<div class="column half_size">
+
<li>Transfer 100 µL of Fluorescein stock solution from A1 into A2</li>
−
<h5>Inspiration</h5>
+
<li>Mix A2 by pipetting up and down 3x and transfer 100 µL into A3
 +
Repeat the process for A3 into A4, A4 into A5, etc. until A11</li>
 +
<li>Mix A11 by pipetting up and down 3x and transfer 100 µL into liquid waste</li>
 +
<li>Repeat dilution series for rows B, C and D</li>
 +
<li>Measure fluorescence of all samples in all standard measurement modes in
 +
instrument</li>
 +
<li>Record the data</li></ul>
 +
<br>
 +
Measurement notes
 +
<ul><li>The plates can now be measured in the plate reader</li>
 +
<li>Standard GFP settings must be used (same as those used when measuring the
 +
cells):<ul>
 +
<li>Excitation 485nm
 +
<li>Emission 530/30
 +
<li>Turn off path length correction</li></ul></li>
 +
<li>Would be ideal to repeat measurements with different settings
 +
<ul><li>Generates series of standard curves to choose from</li></ul></li>
 +
<li>Use number of settings that affect sensitivity (gain and/or slit width)
 +
<ul><li>Also consider orbital averaging, top/bottom optics</li></ul></li>
 +
 
 +
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb13" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb13">Cell Measurement Protocol</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
The calibration measurements should be performed before the measurements on the cells are performed. This allows that the measurement process is understood
 +
and that the cell measurements are taken under the same conditions.
 +
<br>
 +
Materials
 
<ul>
 
<ul>
−
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
+
<li>Competent cells (E.coli strain DH5-alpha)</li>
−
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
+
<li>LB (Luria Bertani) media</li>
−
<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
+
<li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH –
 +
working stock 25 ug/mL)</li>
 +
<li>50 mL Falcon tube (covered in foil to block light)</li>
 +
<li>Incubator at 37oC</li>
 +
<li>1.5mL Eppendorf tubes for sample storage</li>
 +
<li>Ice bucket</li>
 +
<li>Pipettes</li>
 +
<li>96 well plate (black with flat, transparent/clear bottom)</li>************??????
 
</ul>
 
</ul>
 +
 
</div>
 
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb14" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb14">Calcium Chloride Competent Cells</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
Prior Preparation
 +
<ul><li>Autoclave 50mM Calcium Chloride and keep it cold at about 4 o C</li>
 +
<li>For the starter cultures<ul><li>
 +
<li>Add a colony of E.coli DH5cells to 5mL of LB</li>
 +
<li>Incubate at 37 o C overnight</li></ul></li>
 +
<br>
 +
Method:<ul>
 +
<li> Keep cells on ice at all times where possible</li>
 +
<li> To 100mLs of LB, add 100uL of cells from the overnight culture</li>
 +
<li> Let it grow at 37 o C and 250 rpm (until it reaches OD 600 ~0.6-0.8)</li>
 +
<li> Place cells on ice immediately to cool them once the correct OD 600 has been
 +
reached</li>
 +
<li>Centrifuge at max speed for 10 mins and 4 o C</li>
 +
<li>Discard supernatant</li>
 +
<li>Resuspend the pellet in 50% of the original volume with ice-cold 50mM CaCl 2; In a 5omL culture, add 25mL CaCl 2</li>
 +
<li>Allow them to sit on ice for 30 mins</li>
 +
<li>Centrifuge at max speed for 10 mins at 4 o C</li>
 +
<li>Discard the supernatant</li>
 +
</ul>
 +
<br>
 +
<div class="lineSeparator"></div>
 +
<br>
 +
Preparation of Competent Cells for Storage
 +
<br>
 +
<br>
 +
Materials
 +
<ul>
 +
<li>Cell Line</li>
 +
<li>Sterile LB</li>
 +
<li>10mM sterile and chilled Calcium Chloride</li>
 +
<li>Dry ice</li>
 +
<li>Acetone</li></ul>
 +
<br>
 +
Method
 +
<ul>
 +
<li>Inoculate the cells (either 1:50 or 1:100) into 50mL of LB</li>
 +
<li>Grow them at 37 o C until OD600 is around 0.4-0.5</li>
 +
<li>Place on ice for 10 minutes while Falcon tubes are pre-chilled</li>
 +
<li>The cells should be harvested at 3000 rpm, 4C for 8 minutes</li>
 +
<li>The pellet then needs to be resuspended in 1mL of 100mM CaCl 2 and 30%
 +
(v/v) glycerol</li>
 +
<li>The resulting solution needs to be aliquoted into chilled Eppendorf tubes
 +
(100uL per tube)</li>
 +
<li>Place each Eppendorf tube into an acetone dry ice bath to snap freeze them</li>
 +
<li>Then store at -80 o C</li></ul>
  
 +
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
  
−
<div class="clear"></div>
+
</nav>
 +
<div class="connector">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/b/bf/T--Kent--ExperimentsConnect.png/133px-T--Kent--ExperimentsConnect.png">
 +
</div>
 +
<nav class="droptext arrows">
 +
<header class="hull">
 +
<label for="acc-close" class="hull-title">Complex Protocols</label>
 +
</header>
 +
<input type="radio" name="droptext" id="cb15" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb15">DNA Miniprep Kit (Qiagen)</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
Method: (passive + our)
 +
<ul><li>2 x 5 mL of our ampicillin resistant bacteria, containing the plasmid of interest and grown
 +
overnight on LB medium, are centrifuged in falcon tubes at 4500 rpm for 6 minutes.</li>
 +
<li>The supernatant is removed and the pelleted bacteria are resuspended in 250μL of P1 buffer
 +
(containing 100 μg/mL RNase A). Thoroughly mix/ Vortex mix of the samples is required to
 +
ensure full resuspension. The samples are transferred into Eppendorf tubes.</li>
 +
<li>250 μL of P2 buffer is added to each sample and gently mixed by inverting the tube ca. 10 times.
 +
This lysis reaction should not exceed 5 minutes.</li>
 +
<li>350 μL of N3 buffer is pipetted to each sample, and gently but thoroughly mixed by inverting the
 +
tube ca. 10 times. The samples are then centrifuged in a table top centrifuge at 13.000 rpm for
 +
10 minutes.</li>
 +
<li>The supernatant contains our plasmid of interest, while the white pellet is cell debris. 800 μl of
 +
the supernatant are pipetted into Qiagen Spin Columns.</li>
 +
<li>The columns are centrifuged for 60 seconds. The plasmids are retained in a silica mesh, while
 +
remaining substances flow through the column into a collection tube.</li>
 +
<li>The column is washed with 500 μL of PB buffer and centrifuged (13.000 rpm for 60 sec) to
 +
remove any remaining nucleases which could interfere with further processing of the plasmids.</li>
 +
<li>750 μL of PE buffer is added to each sample and centrifuged for 60 seconds to remove any
 +
remaining wash buffer. The flow through is discarded and the spin column is placed into a fresh
 +
Eppendorf tube.</li>
 +
<li>To elute the bound plasmid DNA, 50 μL of EB buffer is added to the column. After letting the
 +
samples stand for ca. 2 minutes, each tube is centrifuged at high speed (13.000 rpm) for 60
 +
seconds.</li>
 +
<li>The spin column is discarded, the Eppendorf tubes now contain our desired plasmid DNA.</li></ul>
 +
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb16" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb16">Enzyme Digest Protocol</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">
 +
A restriction enzyme digestion is usually performed in a volume of 20μL with 0.2-1.5μg of substrate DNA and two-to tenfold excess of enzyme.
 +
<br>
 +
If a large volume of DNA or enzyme is used, abnormal results may occur
 +
<br>
 +
When pipetting the samples in the different lanes of the gel, the enzyme componentof the tube needs to make up 1μL.
 +
<br>
 +
Method:
 +
1. The 5 lanes of the gel are as follows<ul>
 +
<li>Marker</li>
 +
<li>Control (with no cutting enzyme)</li>
 +
<li>1μL EcoR1</li>
 +
<li>1μL Pst1</li>
 +
<li>1μL EcoR1 and Pst1</li>
 +
2. Assemble the following components in a sterile tube:
 +
<br><br>
 +
<div class="TableBox">
 +
<img src="https://static.igem.org/mediawiki/2017/thumb/8/8d/T--Kent--EnzymeDigest.png/800px-T--Kent--EnzymeDigest.png" id="EnzymeTable">
 +
<br><br>
 +
</div>
 +
Note: Different lanes require different tubes to be made up
 +
3. Mix the solution gently by pipetting up and down
 +
4. Close the tube and centrifuge for a few seconds in a microcentrifuge
 +
5. Incubate at the specific enzyme’s optimum temperature (37 o C in this case)for 1-4 hours
 +
6. Add loading buffer to a 1 X final concentration and proceed to the gel analysis
 +
</div>
 +
</section>
 +
<input type="radio" name="droptext" id="acc-close" />
 +
<input type="radio" name="droptext" id="cb17" />
 +
<section class="hull">
 +
<label class="hull-title" for="cb17">PCR Protocol for Q5 High-Fidelity 2X Master Mix</label>
 +
<label class="hull-close" for="acc-close"></label>
 +
<div class="hull-content">All reaction components should be assembled on ice then quickly transferred to a thermocycler that’s been preheated to the denaturation temperature (98oC)
 +
<br>
 +
Components:
 +
All the components should be mixed prior to use
 +
<br><br>
 +
<div class="TableBox"><img src="https://static.igem.org/mediawiki/2017/c/cd/T--Kent--PCR1.png" id="PCR1"></div>
 +
<br><br>
 +
Method:
 +
<ul>
 +
<li>Gently mix the reaction</li>
 +
<li>Collect all the liquid found at the bottom of the tube by a quick spin if needed</li>
 +
<li>Overlay the sample with mineral oil when using a PCR machine that doesn’t have a heated lid</li>
 +
<li>Transfer the PCR tubes to the PCR machine to begin thermocycling</li></ul>
 +
<br>
 +
Thermocycling conditions:
 +
<br><br>
 +
<div class="TableBox"><img src="https://static.igem.org/mediawiki/2017/9/91/T--Kent--PCR2.png" id="PCR2"></div>
 +
<br><br>
 +
Annealing temperatures shouldn’t exceed 72 o C. You can use the NEB T m Calculator
 +
found on the New England BioLabs website to calculate temperatures needed and
 +
timings.
 +
<br>
 +
<br>
 +
<div class="lineSeparator"></div>
 +
<br>
 +
Guidelines
  
 +
Template
 +
<ul><li>A high quality, purified DNA template is preferred as it greatly improves PCR success. Recommended amounts of such a template are shown below for a 50uL reaction:</ul></li>
 +
<div class="TableBox"><img src="https://static.igem.org/mediawiki/2017/thumb/f/f3/T--Kent--PCR3.png/800px-T--Kent--PCR3.png" id="PCR3"></div>
  
−
<div class="column half_size">
+
<br>
 +
<br>
 +
Primers
 +
<ul><li>Oligonucleotide primers should generally be 20-40 nucleotides long while having a GC content of 40-60%</li>
 +
<li>Best results are seen when using each primer at a final concentration of 0.5uM in the reaction</li></ul>
  
 +
<br>
 +
Mg2+ and additives
 +
<ul><li>The Q5 High-Fidelity Master Mix contains 2mM Mg++ when used at a 1X concentration, which is optimal for most PCR products</li></ul>
  
 +
<br>
 +
Deoxynucleotides
 +
<ul><li>Final concentration of dNTPs is 200uM of each deoxynucleotide in the 1X final concentration</li>
 +
<li>Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and isn’t recommended for use with uracil-containing primers or templates</li></ul>
 +
 +
<br>
 +
Q5 High-Fidelity DNA Polymerase concentration
 +
<ul><li>Concentration in the Master Mix has been optimized for best results under a wide conditions range</li></ul>
 +
 +
<br>
 +
Denaturation
 +
<ul><li>Initial denaturation of 30 seconds occurs at 98oC, which is enough for most amplicons from pure DNA templates.</li>
 +
<li>Though longer denaturation times going up to 3 minutes can be used for templates that require it</li></ul>
 +
 +
<Br>
 +
Annealing
 +
<ul><li>Optimal annealing temperatures for this Master Mix tend to be higher than for other PCR polymerases</li>
 +
<li>Typically 10-30 second annealing steps should be used at 3oC above the Tm of the lower Tm primer</li>
 +
<li>Temperature gradients can also be used to optimize the annealing temperature for each primer pair<ul>
 +
<li>For higher Tm primer pairs, two-step cycling without a separate annealing step can be used</li></ul></li></ul>
 +
 +
<br>
 +
Extension
 +
<ul><li>Recommended extension temperature is 72oC
 +
<ul><li>With the recommended time being between 20-30 seconds per kb for complex, genomic samples.<li></ul><li>
 +
<li>The time can be reduced to 10 seconds per kb for simpler templates (plasmid, E.coli, etc.) or complex templates smaller than 1kb</li>
 +
<li>The extension time can be increased to 40 seconds per kb for cDNA or other long, complex templates if needed</li>
 +
<li>A final extension of 2 minutes at 72oC is recommended</li></ul>
 +
 +
 +
Cycle Number
 +
<ul><li>25-35 cycles yield sufficient products generally</li>
 +
<li>For genomic amplicons, 30-35 cycles are advised</li></ul>
 +
 +
<br>
 +
2-step PCR
 +
<ul><li>Used when primers have annealing temperatures exceeding or are equal to 72oC (≥ 72°C).</li>
 +
<li>This 2-step thermocycling protocol combines annealing and extension into one step</li></ul>
 +
 +
<br>
 +
Amplification of long products
 +
<ul><li>When amplifying products > 6kb, you can increase the extension time to 40-50 seconds per kb.</li></ul>
 +
 +
<br>
 +
PCR Product
 +
<ul><li>Products generated using this Master Mix have blunt ends</li>
 +
<li>If clonding is the next step then blunt-end cloning isn’t recommended</li>
 +
<li>If T/A-cloning is to be done, the DNA should be purified prior to A-addition, since the Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated</li></ul>
  
 
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Latest revision as of 03:52, 2 November 2017


Experiments & Protocols