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| | <input type="radio" name="droptext" id="cb8" /> | | <input type="radio" name="droptext" id="cb8" /> |
| | <section class="hull"> | | <section class="hull"> |
| − | <label class="hull-title" for="cb8">Running Agarose Gel</label> | + | <label class="hull-title" for="cb8">Transformation Guidelines (QuickChange Protocol)</label> |
| | <label class="hull-close" for="acc-close"></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. | + | <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> | | <br> |
| − | <ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a | + | Storage Conditions: |
| − | 250ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
| + | <ul><li>XL1-Blue supercompetent cells should be stored at the bottom of a -80 o C freezer</li> |
| − | seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
| + | <li>They should be placed at -80 o C directly from dry ice shipping container</li> |
| − | 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> | + | Aliquoting Cells |
| − | <li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use | + | <li>Keep the XL1-Blue supercompetent cells on ice at all times</li> |
| − | a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
| + | <li>Essential that BD-Falcon polypropylene tubes are places on ice before cells are |
| − | <li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and | + | thawed</li> |
| − | solidify (maximum 30 mins)</li>
| + | <li>Cells must be aliquoted directly into prechilled tubes (Use of 14-mL BD Falcon Polypropylene Round-Bottom Tubes)</li> |
| − | <li>When the gel has set, remove the comb from the tank (gently!) and then cover the whole | + | <li>These tubes must be used (BD Biosciences Catalog #352059) for the transformation |
| − | tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
| + | protocol</li> |
| − | <li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a | + | <li>The heat-pulse steps’ duration is critical and has been optimized for the thickness as |
| − | tube of this and load the whole tube) into well 1( left hand side) and then choose what you
| + | well as shape of these tubes</li> |
| − | load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
| + | <br> |
| − | <li>Load all of your digests into the wells 2,3, and 4.</li> | + | Length of Heat Pulse |
| − | <li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps | + | <li>Optimal efficiencies observed when cells are heat pulsed for 45 seconds</li> |
| − | don’t matter.</li>
| + | <li>Heat pulsing for at least 45 seconds is recommended, allowing for slight variations |
| − | <li>Once the visible markers have reached the half way point of the tank, turn off the power | + | in incubation length</li> |
| − | supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
| + | <li>Efficiencies noted to decrease sharply when pulsed for <30 seconds or >45 seconds</li> |
| − | UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
| + | <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> |
| | + | |
| | + | <li>The IPTG must be prepared in sterile dH2O </li> |
| | + | <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> | | </section> |
| | <input type="radio" name="droptext" id="acc-close" /> | | <input type="radio" name="droptext" id="acc-close" /> |
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| | </section> | | </section> |
| | <input type="radio" name="droptext" id="acc-close" /> | | <input type="radio" name="droptext" id="acc-close" /> |
| − | <input type="radio" name="droptext" id="cb6" />
| + | |
| − | <section class="hull">
| + | |
| − | <label class="hull-title" for="cb6">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="cb7" />
| + | |
| − | <section class="hull">
| + | |
| − | <label class="hull-title" for="cb7">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="cb8" />
| + | |
| − | <section class="hull">
| + | |
| − | <label class="hull-title" for="cb8">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" />
| + | |
| | </nav> | | </nav> |
| | | | |
Experiments & Protocols
