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+ #PCR1{
+ width:50%;
+ display:inline-block;
+ }
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<section class="hull">
<section class="hull">
− <label class="hull-title" for="cb17">Fluorescein Fluorescence Standard Curve</label>
+ <label class="hull-title" for="cb17">PCR Protocol for Q5 High-Fidelity 2X Master Mix </label>
<label class="hull-close" for="acc-close"></label>
<label class="hull-close" for="acc-close"></label>
− <div class="hull-content">
+ <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)
− 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>
+ 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:
+ <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>
<br>
<br>
− Materials
+ Thermocycling conditions :
− <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>
+
−
+
− <li>Transfer 100 µL of Fluorescein stock solution from A1 into A2</li>
+
− <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>
</div>
Production of Lysogeny broth (LB)
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.
Production of SOB medium and magnesium stock
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.
Production of SOC medium and glucose stock
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.
Production of Glycerol stock
If you wish to store bacteria long term, you will need to create a Glycerol Stock after
inoculating an overnight liquid culture
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
The glycerol stock should then be frozen at -80 o C
Successive freeze and thaw cycles will reduce the stocks shelf life
Running Agarose Gel
After the cells have been miniprepped and the plasmid put through a restriction digest, the agarose gel can be run.
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.
Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)
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)
Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
solidify (maximum 30 mins)
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.
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!)
Load all of your digests into the wells 2,3, and 4.
Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
don’t matter.
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.
Overnights protocol
After a transformation has been run and plates have been streaked and patched, overnight cultures will need to be made:
Add 10mL of LB broth (not agar) into as many autoclaved conical flasks as
needed
Add 10uL of Chloramphenicol into each conical flask as well
Using a pipette tip, scrape up some of the cell colonies on the agar plates
prepared beforehand and drop it into the conical flask
Cover up the flask using aluminum foil
Incubate the cultures at 37oC and 180 rpm
Protocol for transformation/ heat shock
This requires chemically competent cells to be made beforehand. These must be stored at -80
degrees Celsius:
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.
100ul of the chemically competent cells are then pipetted (using the chilled pipette tip) into
a chilled Eppendorf tube.
5ul of DNA is the pipetted into the tube with a chilled tip. This tube is then stored on ice for
30 minutes.
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.
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.
The tube is then incubated at 37 degrees Celsius for 45 minutes. The tube should not be
shaken at all at this point.
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.
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.
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.
Incubate in a 37-degree Celsius incubator for 16-18 hours.
Any colonies that result from this should be plated on a patch plate.
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.
Transformation Guidelines (QuickChange Protocol)
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
Storage Conditions:
XL1-Blue supercompetent cells should be stored at the bottom of a -80 o C freezer
They should be placed at -80 o C directly from dry ice shipping container
Keep the XL1-Blue supercompetent cells on ice at all times
Essential that BD-Falcon polypropylene tubes are places on ice before cells are
thawed
Cells must be aliquoted directly into prechilled tubes (Use of 14-mL BD Falcon Polypropylene Round-Bottom Tubes)
These tubes must be used (BD Biosciences Catalog #352059) for the transformation
protocol
The heat-pulse steps’ duration is critical and has been optimized for the thickness as
well as shape of these tubes
Optimal efficiencies observed when cells are heat pulsed for 45 seconds
Heat pulsing for at least 45 seconds is recommended, allowing for slight variations
in incubation length
Efficiencies noted to decrease sharply when pulsed for <30 seconds or >45 seconds
This defined window of highest efficiency for the XL1-Blue cells results from heat
pulse in step 3 of transformation protocol
To the LB agar, add 80 µg/ml of 5-bromo- 4-chloro- 3-indolyl- β-D-galactopyranoside (X-gal)
20 mM isopropy-1- thio β-D galactopyranoside (IPTG)
Appropriate antibiotic
These are all added to prepare the LB agar plates for blue-white colour screening
Alternatively 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
The IPTG must be prepared in sterile dH2O
The X-gal must be prepared in dimethylformamide (DMF)
IPTG and X-gal MUST NOT be mixed before being pipetted onto the plates since the
chemicals may precipitate.
Experiments & Protocols
