Difference between revisions of "Team:Kent/Experiments"

Revision as of 18:20, 29 October 2017

Experiments & Protocols

Basic Protocols

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.
When making the LB we also made another litre batch and added 15g of agar extract to be able to grow bacteria on plates.

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.
10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm filter sterilize was then used

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.
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.

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.

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.

Kit Protocols

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.
When making the LB we also made another litre batch and added 15g of agar extract to be able to grow bacteria on plates.

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.
10ml of 2M Mg 2+ stock was then added and then brought to 100ml with millipure water, 0.2mm filter sterilize was then used

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.
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.

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.

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.

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.

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.

@@ Line 593: / Line 593: @@
 			<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>
@@ Line 608: / Line 603: @@
 		</section>
 <input type="radio" name="droptext" id="acc-close" />
-<input type="radio" name="droptext" id="acc-close" />
+ <input type="radio" name="droptext" id="cb7" />
-<input type="radio" name="droptext" id="cb7" />
 		<section class="hull">
-			<label class="hull-title" for="cb7">Running Agarose Gel</label>
+			<label class="hull-title" for="cb7">Protocol for transformation/ heat shock</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">
+This requires chemically competent cells to be made beforehand. These must be stored at -80
+degrees Celsius:
 <br>
-<ul><li>Make up some agarose. This is done by taking 0.5g of agarose powder and putting it in a
+<ul><li>First, the competent cells and the plasmid intended for transformation must be thawed on
-ml sterile conical flask, with 50ml of TAE buffer, then microwaving it in small pulses (20
+ice. Additionally, some 1.5ml Eppendorf tubes should be chilled on ice, along with some
-seconds then swirling it around) until it is dissolved. Don’t overheat it or it will evaporate too
+pipette tips.</li>
-much. Make up the evaporated volume to 50ml with distilled water.</li>
+<li>100ul of the chemically competent cells are then pipetted (using the chilled pipette tip) into
-<li>Add 1 vial of cybersafe (ask technical services for a tube of it and add all of it)</li>
+a chilled Eppendorf tube.</li>
-<li>Line the white sides of the tank with the agarose solution, to seal it and prevent leakage. Use
+<li>5ul of DNA is the pipetted into the tube with a chilled tip. This tube is then stored on ice for
-a p1000 pipette set to 1ml. Let it dry (about 5 mins max)</li>
+minutes.</li>
-<li>Then pour all the agarose/sybrsafe solution into the tank and put in the comb. Let it set and
+<li>The tube is then placed in a water bath at 42 degrees Celsius for precisely 90 seconds. After
-solidify (maximum 30 mins)</li>
+seconds is up, the tube is transferred back to ice for 2 minutes.</li>
-<li>When the gel has set, remove the comb from the tank (gently!) and then cover the whole
+<li>900ul SOC medium is then added into the tube (with a normal pipette tip, doesn’t need to
-tank with TAE buffer, so there’s at least half a centimetre of TAE covering the gel.</li>
+be chilled) and then mix very gently by pipetting up and down inside the tube.</li>
-<li>Now, the samples need to be loaded. Load some DNA markers (ask technical services for a
+<li>The tube is then incubated at 37 degrees Celsius for 45 minutes. The tube should not be
-tube of this and load the whole tube) into well 1( left hand side) and then choose what you
+shaken at all at this point.</li>
-load into wells 2, 3, and 4 etc. (make sure you note what’s in each lane!)</li>
+<li>100ul of the transformation mix is then pipetted into the centre of a plate containing LB agar
-<li>Load all of your digests into the wells 2,3, and 4.</li>
+and the appropriate antibiotic (for example, for plasmid pSB1C3, Chlorophenicol should be
-<li>Plug into a power supply and put the cover on. Run for 40 mins to an hour at 80v. The amps
+used, and for pSB1A3, use Ampicillin). Use 1ul of antibiotic for each ml of agar.</li>
-don’t matter.</li>
+<li>In sterile conditions (Bunsen burner, gloves cleaned with IMS (ALLOWED TO DRY)), spread
-<li>Once the visible markers have reached the half way point of the tank, turn off the power
+the bacteria around the plate by keeping the lid as closed as possible and inserting the
-supply and drain the TAE buffer form the tank. Remove the gel with a spatula and place in a
+spreader then turning the plate around to spread the cells. Then immediately close and
-UV imaging box. Take an image of the gel under UV light, save it onto a USB stick.</li></ul></div>
+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" />