Team:Tec-Monterrey/ITESM14 protocols.html

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Here you can find a detailed description of the different experimental protocols that were used for the making of our project.

<a data-toggle="collapse" data-parent="#accordion" href="#collapse1"> Protocol 1-Calcium competent cells </a>

Approximate time
Around 5 hours (depends in the growth rate of the strain)
Material
100 mM CaCl₂
200mL LB
4 sterile centrifuge tubes
Overnight E. coli culture
Equipment & Apparatus
Centrifuge (Thermo)
Spectrometer (Thermo)
Laminar flow hood (Scientific Visions)
Incubator with shaker (TalBoys)
Previous steps
Autoclave. 200mL of LB media placed in a 1L flask, Glycerol, 4 centrifuge tubes, microcentrifuge tubes, 1L of 100 mM CaCl₂.
Chill overnight at 4°C. 100 mM CaCl₂.
Prepare starter culture cells. Inoculate a 25mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.
Steps
1.	Inoculate 200mL of LB media with approximately 20mL starter culture. Measure the OD600 to make sure it is around 0.1
2.	Grow in 37°C shaker.
3.	Measure the OD₆₀₀ every 30 minutes. When the OD₆₀₀ reaches 0.4-0.6, immediately put the cells on ice. Chill the culture for 30 minutes.
4.	Harvest the cells by centrifugation at ~4000 rpm for 15 minutes at 4°C.
5.	Decant the supernatant and gently resuspend each pellet in about 30mL of ice cold MgCl₂.
6.	Put the cells on ice and chill the culture for 30 minutes.
7.	Repeat two times the steps 4-6 varying the incubation time by 20 and 15 minutes respectively.
8.	Harvest the cells by centrifugation at ~4000 rpm for 15 minutes at 4°C.
9.	Decant the supernatant and gently resuspend the pellet of one of the tubes in 500μL of ice cold MgCl₂ and 15% of glycerol.
10.	Once the pellet is completely resuspended, transfer the solution into the next tube and resuspend the pellet. Repeat this step for all the pellets.
11.	Aliquot 50μL into sterile 1.5 mL microfuge tubes and store at -80°C freezer.
Waste disposal
Waste	Place to dispose	What to avoid
LB medium	Place in a container with chlorine solution	N/A
CaCl solution	Place in a container with chlorine solution	N/A
Tips	Place in a container and with chlorine solution then sterilize	Be ordered and use the container all the time
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

<a data-toggle="collapse" data-parent="#accordion" href="#collapse2"> Protocol 2-Transformation of Calcium Competent Cells </a>

Approximate time
90 min
Material
Liquid LB media
Petri dishes
Equipment & Apparatus
Incubator with shaker (TalBoys)
Laminar flow hood (Scientific Visions)
Water bath (Thermo)
Previous steps
Autoclave. LB liquid media, LB-agar media.
Steps
1.	Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 50μL of competent cells in a microcentrifuge tube.
2.	Place the competent cell/DNA mixture on ice for 20min.
3.	Heat shock each transformation tube by placing the bottom of the tube into a 42°C water bath for 45 seconds (time varies depending on the competent cells you are using).
4.	Put the tubes back on ice for 2 min.
5.	Add 500μl LB media (without antibiotic) and grow in 37°C shaking incubator for 1 hour.
6.	Plate 100 μl of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
7.	Incubate plates at 37°C overnight.
Waste disposal
Waste	Place to dispose	What to avoid
Petri dishes with culture	Place in a bag to sterilize	N/A
Liquid LB with culture	Place in a container and with chlorine solution then sterilize	N/A
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). “Basic Protocol 1: Transformation Using Calcium Chloride.” Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

<a data-toggle="collapse" data-parent="#accordion" href="#collapse3"> Protocol 3-Miniprep </a>

Objective
To isolate a plasmid from an E. coli culture
Approximate time
60-180 min (depends in the incubation time)
Material
Solution I. (50 mM TRIS pH 8.0, 10 mM EDTA)
Solution II (200 mM NaOH, 1% w/v SDS)
Solution III (3 M potassium acetate, pH 5.5)
EtOH 100%
EtOH 70%
ddH₂O
Equipment & Apparatus
Nanodrop (Thermo)
Centrifuge (BioRad)
Previous steps
Prepare starter culture cells. Inoculate a 15mL starter culture of LB with the cells that have the desired plasmid (with the appropriate antibiotic). Grow culture at 37°C in shaker overnight.
Steps
1.	Centrifuge the starter culture.
2.	Decant the supernatant and resuspend the pellet in about 1mL of LB media
3.	Transfer the cells into a Eppendorf tube.
4.	Centrifuge 14,000 rpm / 1 minute.
5.	Decant the supernatant.
6.	Add 200uL of solution I.
7.	Use a pipette to resuspend the pellet.
8.	Add 2uL of RNAse.
9.	Incubate 5min at room temperature.
10.	Add 200uL of Solution II.
11.	Mix gently by inversion 6 times.
12.	Incubate 5 min at room temperature.
13.	Add 200uL of Solution III.
14.	Mix gently by inversion 6 times.
15.	Incubate 5 min on ice.
16.	Centrifuge 14,000 rpm for 10 min.
17.	Transfer the supernatant to a new tube (around 500uL).
18.	Add 2 volumes cool of 100% EtOH (around 1mL).
19.	Incubate at -20°C for 10 min (can be from 10 min to 2 hours).
20.	Centrifuge 14,000 rpm for 10 min.
21.	Decant the supernatant.
22.	Add 200uL of cool 70% EtOH.
23.	Use a pipette to resuspend the pellet.
24.	Centrifuge 14,000 rpm for 5 min.
25.	Decant the supernatant.
26.	Dry pellet at 37 ° C for 5 min.
27.	Add 30uL of ddH₂O and resuspend pellet.
28.	Measure DNA concentration in nanodrop.
Waste disposal
Waste	Place to dispose	What to avoid
Tube with cell pellets	Pour chlorine solution and clean the tube	N/A
Eppendorf with protein waste	Dispose the tube in normal container	N/A
Safety Notes
Gloves must be worn at all times Can be done safely outside the hood Try to keep the tubes closed to avoid contamination
References


Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

<a data-toggle="collapse" data-parent="#accordion" href="#collapse4"> Protocol 4-Electro-competent cells </a>

Approximate time
Around 5 hours (depends in the growth rate of the strain)
Material
Sterile dH₂O
200mL LB
4 sterile centrifuge tubes
Overnight E. coli culture
Equipment & Apparatus
Centrifuge (Thermo)
Spectrometer (Thermo)
Laminar flow hood (Scientific Visions)
Incubator with shaker (TalBoys)
Previous steps
Autoclave. 200mL of LB media placed in a 1L flask, Glycerol, 4 centrifuge tubes, microcentrifuge tubes, 1L of sterile dH₂O.
Chill overnight at 4°C. 100 mM CaCl₂.
Prepare starter culture cells. Inoculate a 25mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.
Steps
1.	Inoculate 200mL of LB media with approximately 20mL starter culture. Measure the OD₆₀₀ to make sure it is around 0.1
2.	Grow in 37°C shaker.
3.	Measure the OD₆₀₀ every 30 minutes. When the OD₆₀₀ reaches 0.4-0.6, immediately put the cells on ice. Chill the culture for 30 minutes.
4.	Harvest the cells by centrifugation at ~4000 rpm for 15 minutes at 4°C.
5.	Decant the supernatant and gently resuspend each pellet in about 30mL of ice cold dH₂O.
6.	Put the cells on ice and chill the culture for 30 minutes.
7.	Repeat two times the steps 4-6 varying the incubation time by 20 and 15 minutes respectively.
8.	Harvest the cells by centrifugation at ~4000 rpm for 15 minutes at 4°C.
9.	Decant the supernatant and gently resuspend the pellet of one of the tubes in 500μL of ice cold dH₂O and 15% of glycerol.
10.	Once the pellet is completely resuspended, transfer the solution into the next tube and resuspend the pellet. Repeat this step for all the pellets.
11.	Aliquot 50μL into sterile 1.5 mL microfuge tubes and store at -80°C freezer.
Waste disposal
Waste	Place to dispose	What to avoid
LB medium	Place in a container with chlorine solution	N/A
dH₂O	Place in a container with chlorine solution	N/A
Tips	Place in a container and with chlorine solution then sterilize	Be ordered and use the container all the time
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

<a data-toggle="collapse" data-parent="#accordion" href="#collapse5"> Protocol 5-Transformation of electro-competent cells </a>

Approximate time
Around 2 hours
Material
1 mL, 100µL and 10µL Micropipettes
Liquid LB media
Petri dishes
Electroporation cells
Equipment & Apparatus
Incubator with shaker (TalBoys)
Laminar flow hood (Scientific Visions)
Electroporator (BioRad)
Previous steps
Autoclave. LB liquid media (500 µL per transformation), LB-agar media (one control per antibiotic).
Steps
1.	In the flow hood, place 1 µL of DNA in one side of the cell.
2.	Using the pipette pour 50 µL of the competent cells in the same side of the electroporation cell, so the DNA and cells will mix and reach the bottom
3.	Electroporate the cell outside the hood using a potential of 1.8 mV
4.	If the transformation is successful, a curve will appear on the screen of the electroporator otherwise an “arc” sign will appear.
5.	Using the micropipette, place 500 µL of LB media in the cell.
6.	Add 500μl LB media (without antibiotic) and grow in 37°C shaking incubator for 1 hour.
7.	Plate 100 μl of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
8.	Incubate plates at 37°C overnight.
Waste disposal
Waste	Place to dispose	What to avoid
Petri dishes with culture	Place in a bag to sterilize	N/A
Liquid LB with culture	Place in a container and with chlorine solution then sterilize	N/A
Tips	Place in a container and with chlorine solution then sterilize	Be ordered and use the container all the time
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
Seidman, C. E., Struhl, K., Sheen, J., & Jessen, T. (2005). Introduction of plasmid DNA into cells. Current protocols in molecular biology, 1-8.

Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

<a data-toggle="collapse" data-parent="#accordion" href="#collapse6"> Protocol 6-Digestion with restriction enzymes </a>

Objective

Analyze DNA constructs and assemblies using restriction enzymes.

Cut DNA fragments in order to ligate them.

Approximate time

2 hrs

Material

PCR tubes

DNA sample

Restryction Enzymes (EcoRI, SpeI, PstI, XbaI)

NEB Enzyme buffer

Nuclease free dH₂O

Equipment & Apparatus

Thermocycler (BioRad)

Nanodrop (Thermo)

Previous steps

Measure the DNA concentration of the sample using the Nanodrop.

Calculate the required concentrations of the components depending on the DNA of the sample, and the reaction volume.

Ideally you must put 1 enzyme unit per each µg of DNA, yet the provider recommends using 10 times that quantity and adjust the volumes. Our most typical reaction was set up to be 20 µL.

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Reaction volume	Restriction Enzyme	DNA	10X BEBuffer
10 µl rxn	1 unit	0.1 µg	1 µl
25 µl rxn	5 units	0.5 µg	2.5 µl
50 µl rxn	10 units	1 µg	5 µl

Steps

1.

Set and label your PCR tubes.

2.

With the micropipette first pour the nuclease free dH₂O.

3.

Pour the NEB buffer 10X to get the 1X concentration.

4.

Put the DNA sample next, this sample must be as clean as possible.

5.

The enzymes must be put lastly, be careful to handle them because they are very temperature sensitive. (Handle with ice and return them to the fridge as soon as possible).

6.

Set up the Thermocycler at 37º for 1h and inactivate the enzyme at 80º for 20 mins.

7.

Analyze using agarose electrophoresis.

Waste disposal

Waste

Place to dispose

What to avoid

Digestion waste in PCR tube

Place in trash container.

N/A

Safety Notes

Gloves must be worn at all times.
Handle the enzymes carefully.
Typically the digestion will be used to confirm or to use the digestion to ligate. The first could generate some waste, but the second usually won't.

References

Double Digest Protocol with Standard Restriction Enzymes <a href="https://www.neb.com/protocols/2014/05/07/double-digest-protocol-with-standard-restriction-enzymes" target="_blank"> https://www.neb.com/protocols/2014/05/07/double-digest-protocol-with-standard-restriction-enzymes</a>

Optimizing Restriction Endonuclease Reactions <a href="https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions" target="_blank"> https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions</a>

Digestion Protocol for BioBrick Assembly Kit (E0546)<a href="https://www.neb.com/protocols/1/01/01/digestion-protocol-e0546" target="_blank"> https://www.neb.com/protocols/1/01/01/digestion-protocol-e0546</a>

Edited by

Eduardo A. Ramírez Rodríguez

Date

September 9th, 2014

Revision

<a data-toggle="collapse" data-parent="#accordion" href="#collapse7"> Protocol 7-Agarose based electrophoresis </a>

<col style="width:8%"> <col style="width:32%"> <col style="width:20%"> <th colspan=3>Related protocols </th> </tr> <tr> <td colspan=3>Digestion, enzyme restriction analysis.</td> </tr> <tr> <th colspan=3>Steps</th> </tr> <tr> <td colspan=3>Prepare the gel </td> </tr> <tr> <td align="center">1.</td> <td colspan=2> The amount of agarose depends on the size of the DNA. (A guide can be seen in the table below) </td> <tr> <td></td> <td colspan=2>

Objective
Analyze DNA fragments based on their movement in a uniform electric field.
Approximate time
120 min
Material
Agarose (BioRad)
TAE Buffer (Promega)
Loading dye (Promega)
10 kb and 1 kb weight stair (Promega)
Ethidium bromide (BioRad)
Pipettes and tips
DNA sample
Equipment & Apparatus
Electrophoresis chamber (Fisher Scientific)
Voltage source (BioRad)
Transiluminator (Thermo)
Previous steps
Digestion. Usually this protocol precedes the electrophoresis.
Check if there is TAE available. A stock 50X can be made from: Tris-base: 242 g </li> Acetate (100% acetic acid): 57.1 ml </li> EDTA: 100 ml 0.5M sodium EDTA </li> Add dH₂O up to one litre. </li>

                   <col style="width:50%">
                   <col style="width:50%">

Agarose Concentration (g/100mL)	Optimal DNA Resolution (kb)
0.5	1 - 30
0.7	0.8 - 12
1.0	0.5 - 10
1.2	0.4 - 7
1.5	0.2 - 3

</td>

                 </tr>
             </tr>
             <tr>
               <td align="center">2.</td> <td colspan=2>Measure out the appropriate mass of agarose into a beaker with the appropriate volume of buffer. The volume in the lab gelbox is 50mL. </td> 
             </tr>
             <tr>
               <td align="center">3.</td> <td colspan=2>Microwave until the agarose is fully melted and cool down a few minutes.</td> 
             </tr>
             <tr>
               <td align="center">4.</td> <td colspan=2> Pour the agarose solution into the taped gelbox. Carefuly pop or shove to the side any bubbles, put in the comb, and let it cool for about 30 minutes, until the gel is solid. </td> 
             </tr>
             <tr>
               <td colspan=3>Load the gel </td> 
             </tr>
             <tr>
               <td align="center">5.</td> <td colspan=2>Mix the DNA samples with 5X loading dye solution to make it reach 1X. (Except for 1 µL of sample you can use 1µL of Loading dye). </td> 
             </tr>
             <tr>
               <td align="center">6.</td> <td colspan=2>Pour the mixture in the wells. Be careful with the bubbles and breaking the gel with the pipette. </td> 
             </tr>
             <tr>
               <td align="center">7.</td> <td colspan=2>Fill the chamber with TAE and run the gel 70 min at 90 V and ~180 mA </td> 
             </tr>
             <tr>
               <td colspan=3>Stain and visualize the gel  </td> 
             </tr>
             <tr>
               <td align="center">8.</td> <td colspan=2>Stain the gel soaking it in EtBr, be careful not to break it. </td> 
             </tr>
             <tr>
               <td align="center">9.</td> <td colspan=2>Reveal usin the transiluminator, first focus using the white light lamp and use the UV to make the bands visible. </td> 
             </tr>
           <tr>
           <th colspan=3>Waste disposal</th>
           </tr>
             <tr>
               <td id="tsubtitle">Waste</td> <td id="tsubtitle">Place to dispose</td> <td id="tsubtitle">What to avoid</td> 
             </tr>
             <tr>
               <td>TAE</td> <td>Throw through the sink, the school has a water treatment plant for all the facilities. </td> <td>N/A</td> 
             </tr>
             <tr>
               <td>Gel</td> <td>The gel has EtBr, which is very mutagenic, gloves must be used to handle the gel when you soak it in the solution. The gel must be disposed in the EtBr container</td> <td>Do not touch the gel or the EtBr solution with your bare hands</td> 
             </tr>
             <tr>
               <td>EtBr solution</td> <td>Every once in a while the solution must be changed, and the discarded solution must be</td> <td>See above</td> 
             </tr>
             <tr>
               <td>Gloves </td> <td>You must discard the glove you use when you handle the gel and the computer. They must be thrown in the EtBr labeled container.</td> <td>Make sure to dispose your gloves</td> 
             </tr>
           <tr>
           <th colspan=3>Safety Notes</th>
           </tr>
             <tr>
               <td colspan=3>

Remember to use the EtBr at the delimited area in the lab.
Dispose your gloves after handling the gel.
If you accidentally touch a surface of EtBr designed area, remember to soak yourself with ethanol 70% to inactivate the EtBr. Seriously, this shouldn’t happen.

               </td>
             </tr>
           <tr>
             <th colspan=3>References</th>
           </tr>
             <tr>
               <td colspan=3>Agarose Gel Electrophoresis <a href="http://www.addgene.org/plasmid-protocols/gel-electrophoresis/">http://www.addgene.org/plasmid-protocols/gel-electrophoresis/</a> </td>
             </tr>
               <tr><td colspan=3> </td></tr>
             <tr>
           <td colspan=1 id="tsubtitle2" >Edited by</td><td colspan=2>Eduardo A. Ramírez Rodríguez </td>
           </tr>
           <tr>
             <td id="tsubtitle" colspan=1>Date</td><td colspan=2>September 28th, 2014 </td>
           </tr>
           <tr>
             <td id="tsubtitle" colspan=1>Revision</td><td colspan=2></td>
           </tr>
         </table>
       </center>
     </div>
   </div>
 </div>

<a data-toggle="collapse" data-parent="#accordion" href="#collapse8"> Protocol 8-Cell Lysis and Protein Purification </a>

Objective

Extract the protein produced by E. coli and purification of polyhistidine-tagged proteins from a soluble protein extract by metal affinity chromatography.

Material

500mL solution Tween 20 5% v/v in K₂HPO₄ 25mM pH 7.5.

100mL Biuret reagent

Equilibration Buffer: 10mM Imidazole

Wash Buffer: 25mM Imidazole

Elution Buffer: 250mM Imidazole

Equipment & Apparatus

Incubator with shaker (TalBoys)

Sonicator

Spectrometer (Thermo)

50 and 15mL plastic tubes

Centrifuge (Thermo)

HisPur Ni-NTA Spin Columns

Previous steps

Grow E. coli culture to 0.8OD.Centrifuge and keep the pellet (wet biomass) for lysis. Supernatant can be kept to compare the concentration of protein with the lysate.

Prepare Purification Buffers under this specifications:

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Imidazole Finale Conc. (mM)	10X PBS (mL)	2M Imidazole (µL)	Water (mL)
10	1	50	8.95
25	1	125	8.875
40	1	200	8.8
60	1	300	8.7
75	1	375	8.625
150	1	750	8.25
200	1	1000	8
250	1	1250	7.75
500	1	2250	6.5

Steps

1.

Take 2.0g of wet biomass and place it in a plastic test tube up to 50ml.

2.

Resuspend the solution in 28ml of Tween 20 5% v/v in K₂HPO₄ 25mM pH 7.5.

3.

Introduce the sonication probe in the suspension of cells. Sonicate the sample 10 seconds and turn of another 10 seconds for 3 minutes. The tube should be put in ice during the procedure.

4.

Centrifuge samples (4,000rpm) for 2 min.

5.

Quantify total protein in the lysate and supernatant using Biuret method. Use LB medium as blank with Biuret reagent.Spectrometer reading at 540nm should be compared with standard curve previously made.

Purification

6.

Prepare sample by mixing protein extract with Equilibration Buffer so the total volume equals two resin-bed volumes.

7.

Remove the bottom tab from the HisPur Ni-NTA Spin Column by gently twisting. Place column into a centrifuge tube. Use 1.5, 15 or 50mL centrifuge tubes for the 0.2, 1 and 3mL spin columns, respectively.

8.

Centrifuge column at 700 × g for 2 minutes to remove storage buffer.

9.

Equilibrate column with two resin-bed volumes of Equilibration Buffer. Allow buffer to enter the resin bed.

10.

Centrifuge column at 700 × g for 2 minutes to remove buffer.

11.

Add the prepared protein extract to the column and allow it to enter the resin bed. For maximal binding, the sample can be incubated for 30 minutes at room temperature or 4°C on an end-over-end rocking platform.

12.

Centrifuge column at 700 × g for 2 minutes and collect the flow-through in a centrifuge tube.

13.

Wash resin with two resin-bed volumes of Wash Buffer. Centrifuge at 700 × g for 2 minutes and collect fraction in a centrifuge tube. Repeat this step two more times collecting each fraction in a separate centrifuge tube.

14.

Elute His-tagged proteins from the resin by adding one resin-bed volume of Elution Buffer. Centrifuge at 700 × g for 2 minutes. Repeat this step two more times, collecting each fraction in a separate tube.

Waste disposal

Waste

Place to dispose

What to avoid

LB Medium

Place in container with chlorine solution.

N/A

Supernatant Waste

Sterilize.

N/A

Wash Buffer

Place in container with chlorine solution and sterilize.

N/A

Safety Notes

Gloves must be worn at all times.

References

Thermo Scientific HisPur^TM Ni-NTA Purification Kit<a target="_blank" href="https://www.piercenet.com/instructions/2162206.pdf "> https://www.piercenet.com/instructions/2162206.pdf </a>

Edited by

Eduardo Serna Morales

Date

September 28th, 2014

Revision

<a data-toggle="collapse" data-parent="#accordion" href="#collapse9"> Protocol 9-Red Lambda Protocol (revisar referencias y autor) </a>

Objective
To make target genetic changes in E. coli.
Approximate time
3.5h
Material
25 mL LB
1.1mL glycerol
IPTG
MOPS 1mM
Equipment & Apparatus
Incubator with shaker (TalBoys)
Laminar flow hood (Scientific Visions)
Centrifuge (Thermo)
Electroporation System (Gene Pulser MXcell)
Previous steps
Autoclave. 200mL of LB media placed in a 1L flask, Glycerol, 4 centrifuge tubes, microcentrifuge tubes, 1L of 100 mM CaCl₂.
Related protocols
Autoclave. 200mL of LB media placed in a 1L flask, Glycerol, 4 centrifuge tubes, microcentrifuge tubes, 1L of 100 mM CaCl₂.
Steps
1.	Select a colony and place it on 20mL LB + 100 µg/ml Amp 30°C in a 125mL flask.
2.	Grow up to ~10⁷ cel/mL.
3.	Add IPTG to final concentration of 1mM.
4.	After reaching the density between 0.5-1x10⁸ cel/mL apply heatshock for 15 min at 42°C swirling.
5.	Transfer to ice for 10min (swirling).
6.	Centrifuge (Cell recovery).
7.	Resuspend in 1mL of glycerol 20% - 1mM MOPS (unbuffered) frozen.
8.	Transfer to 1.5mL eppendorf.
9.	Centrifuge 30seg at moderate speed.
10.	Pull supernatant.
11.	Resuspend in the same buffer.
12.	Centrifuge 30seg at moderate speed.
13.	Repeat steps 11 and 12.
14.	Resuspend in 90-100µL 1mM MOPS 20% frozen glycerol.
15.	Cool electroporation cells on ice for 10 mins.
16.	Add 50µL of cells to the electroporation cell, with 1-5 µL of DNA and incubate for 1min on ice.
17.	Dry out the electroporation cell fast and efficiently.
18.	Apply shock. Peak discharge 20kv/cm. RC time 4.2mS. Resistor of 200Ω.
19.	Suspend the cell with 0.3 mL of LB and dilute in 2.7mL of LB.
20.	Grow for 1.5h at 37°C.
21.	Plating with resistance at half.
Waste disposal
Waste	Place to dispose	What to avoid
LB medium	Place in a container with chlorine solution	N/A
Petri dishes with culture	Place in a bag to sterilize.	N/A
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References


Edited by
Date
Revision

<a data-toggle="collapse" data-parent="#accordion" href="#collapse10"> Protocol 10-Quorum Sensing Assay </a>

<col style="width:8%"> <col style="width:32%"> <col style="width:20%"> <tr> <td align="center">6.</td> <td colspan=2>Measure fluorescence of GFP (395nm excitation, 509nm emission, 495nm cutoff). </td> </tr> <tr> <td align="center">7.</td> <td colspan=2>Measure cell density at 600nm. </td> </tr> <tr> <td align="center">8.</td> <td colspan=2>Repeat steps 3 to 7. (For better characterization it is recommended to repeat this steps for 16-20 hours) </td> </tr> <tr> <th colspan=3>Waste disposal</th> </tr> <tr> <td id="tsubtitle">Waste</td> <td id="tsubtitle">Place to dispose</td> <td id="tsubtitle">What to avoid</td> </tr> <tr> <td>LB medium</td> <td>Place in a container with chlorine solution</td> <td>N/A</td> </tr> <tr> <td>Microplate </td> <td>Place in a bag to sterilize. </td> <td>N/A</td> </tr> <tr> <th colspan=3>Safety Notes</th> </tr> <tr> <td colspan=3>

Every step must be done in the hood to avoid contamination
Gloves must be worn at all times

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         <td colspan=1 id="tsubtitle2" >Edited by</td><td colspan=2></td>
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         <td id="tsubtitle" colspan=1>Date</td><td colspan=2></td>
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         <td id="tsubtitle" colspan=1>Revision</td><td colspan=2></td>
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse11"> Protocol 11-Phage Purification by Isoelectric Precipitation </a>

Objective
Characterize the operation of quorum sensing by fluorescence.
Approximate time
Around 5 hours (depends in the growth rate of the strain)
Material
100 mL LB
5mM potassium phosphate buffer
Bioassay medium (0.05% w/v tryptone, 0.03% v/v glycerol, 100mM sodium chloride, 50mM magnesium sulfate and 5mM potassium phosphate buffer, pH 6.5, containing antibiotic)
Overnight E. coli culture
Equipment & Apparatus
Incubator with shaker (TalBoys)
Laminar flow hood (Scientific Visions)
Spectrometer (Thermo)
96-well microplate with clean background
Centrifuge (Thermo)
Previous steps
Autoclave. 100mL of LB , 2 centrifuge tubes.
Prepare starter culture cells. Inoculate a 25mL starter culture of LB (no antibiotics). Grow culture at 37°C in shaker overnight.
Steps
1.	Add overnight to LB medium until OD600nm reaches 0.1
2.	Incubate cell in incubator with shaker (250 rpm and 37°C)
3.	After 2 hours, centrifuge 3mL of sample at 14,000rpm for one minute
4.	Wash the pellet twice with 600µL of PBS 1X.
5.	Load 200µL of the suspension to a 96 wells Costar plate. (Load the suspension in triplicate) Our cells are able to produce acyl-HSL (it is not necessary to add more).</li>

<col style="width:8%"> <col style="width:32%"> <col style="width:20%"> <tr> <td colspan=3>Bacterial cells are removed by centrifugation (5,000 rpm) for 10 min and a second centrifugation (12,000 rpm) for 10 min. </td> </tr> <tr> <th colspan=3>Steps</th> </tr> <tr> <td align="center">1.</td> <td colspan=2>Adjust he pH of the supernatant to pH 4.2 with 5M HCl and is mixed briefly. </td> </tr> <tr> <td align="center">2.</td> <td colspan=2>Concentrate the virus precipitated of the 200mL culture in a 50mL tube by centrifugation (13,000 rpm) for 10min at 20°C. The supernatant is discarded.</td> </tr> <tr> <td align="center">3.</td> <td colspan=2>Remove the residual LB, suspend pellets in 40mL of Mili-Q water by vortexing and centrifuge (13,000 rpm) for 10min at 20°C. </td> </tr> <tr> <td align="center">4.</td> <td colspan=2> Dry washed pellets at 70°C in an oven for calculations of dry weight (covered with a jacket layer Bio-Shield Dupont) or suspend in 10mL Mili-Q water. </td> </tr> <tr> <td align="center">5.</td> <td colspan=2>Adjust the respuspended pellets pH to 7 with 5M NaOH and keep samples at 4°C. </td> </tr> <tr> <th colspan=3>Waste disposal</th> </tr> <tr> <td id="tsubtitle">Waste</td> <td id="tsubtitle">Place to dispose</td> <td id="tsubtitle">What to avoid</td> </tr> <tr> <td>LB medium</td> <td>Place in a container with chlorine solution</td> <td>N/A</td> </tr> <tr> <th colspan=3>Safety Notes</th> </tr> <tr> <td colspan=3>

Every step must be done in the hood to avoid contamination
Gloves must be worn at all times

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         <th colspan=3>References</th>
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           <td colspan=3>Dong, D., Sutaria, S., Hwangbo, J. Y., & Chen, P. (2013). A simple and rapid method to isolate purer M13 phage by isoelectric precipitation. Applied Microbiology and Biotechnology, 97(18), 8023– 9. doi:10.1007/s00253-013-5049-9</td>
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         <td colspan=1 id="tsubtitle2" >Edited by</td><td colspan=2></td>
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         <td id="tsubtitle" colspan=1>Date</td><td colspan=2></td>
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<a data-toggle="collapse" data-parent="#accordion" href="#collapse12"> Protocol 12-Phage Quantification by Spectrophotometry </a>

Objective
To achieve phage recuperation (98.4%) from culture media.
Approximate time
Around 3 hours
Material
250mL of LB medium
5M HCl
50mL of mili-Q water
Overnight E. coli culture
5M NaOH
Equipment & Apparatus
Incubator with shaker (TalBoys)
Laminar flow hood (Scientific Visions)
Centrifuge (Thermo)
Oven for calculations of dry weight
4 tubes for centrifugation
Previous steps
Virus Culture. Add 200µL of E. coli ER2738 and 10µL of virus suspension (approx. 1x1010 PFUml-1) to a suspension of 200mL of LB in an Erlenmeyer flask of 500mL. LB Medium: 10gl^-1 of tryptone. 5gl^-1 NaCl (pH 7.0-7.5). 5gl^-1 of yeast extract. </li> Culture conditions: 37°C 220 rpm 16 h </li>

Objective
To quantify phage concentration in virion/mL.
Approximate time
30 minutes
Material
Milipore DI water
TBS
4 sterile centrifuge tubes
Overnight E. coli culture
Equipment & Apparatus
Nanodrop
Previous steps
Phage purification.
Steps
1.	Start nanodrop with 2µL Milipore DI water.
2.	Use 2µL of 1X TBS as blank for each phage solution at 290nm and 320nm.
3.	To calculate the phage concentration this equation is needed: <figure> <a href="images/protocols/P12.png" data-lightbox="Protocol 12" data-title="Equation 1"><img class="img img-responsive" src="images/protocols/P12.png"></a> </figure>
	If the concentration is above 2OD at 269nm, the preparation is too concentrated; use a bigger dilution factor.
Waste disposal
Waste	Place to dispose	What to avoid
Waste	Wash with detergent and/or hypochlorite solution	N/A
TBS solution	Place in container with chlorine solution.	N/A
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
Yin, H. (2013) Searching for M13 Bacteriophage with affinity for nanodiamond particles. Department of materials science and engineering.

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<a data-toggle="collapse" data-parent="#accordion" href="#collapse14"> Protocol 14-Chromogenic Lal Endotoxin Assay </a>

Objective
To quantifie endotoxins by a modified Limulus Amebocyte Lysate and a synthetic color producing substrate. Detection limit of 0.005 EU/ml, concentration range of 0.005 to 1 EU/ml.
Material
Limulus Amebocyte Lysate (LAL)
Stop Solution
E. coli endotoxin standard
Color standards
0.1N sodium hydroxide
0.1N hydrochloric acid
Equipment & Apparatus
Endotoxin free tubes and tips
Spectrometer (Thermo)
Water Bath (Thermo)
Incubator (Tal Boys)
Previous steps
Dilute test specimen using LAL Reagent Water, adjust pH to 6-8 with 0.1N sodium hydroxide or 0.1N hydrochloric acid.
Prepare 1EU/ml endotoxin solution before making standard serial dilutions. In each assay, at least four endotoxin standard solutions covering desired concentration range should be prepared to generate a standard curve. If the endotoxin concentration for the test sample is expected to be in the range of 0.01 - 0.1 EU/ml, the serial endotoxin standard solutions could be 0.1, 0.05, 0.025 and 0.01 EU/ml, respectively. If the endotoxin concentration in sample is expected in the range of 0.1 - 1 EU/ml, the serial endotoxin standard solutions could be 1, 0.5, 0.25 and 0.1 EU/ml, respectively.
Steps
1.	Dispense 100µL of standard or test sample into endotoxin-free vials. Mix samples for 30 minutes with a vortexer. Each test must include a blank as well as at least four endotoxin standards in duplicate. The blank sample vial contains 100 μl of LAL Reagent Water instead of test sample.
2.	Add 100 µl of reconstituted LAL to each vial. Cap the vials and mix well by swirling gently.
3.	If the endotoxin concentration in sample is expected in the range of 0.01 - 0.1 EU/ml, incubate the rack with all vials at 37°C±1°C for T1 using a water bath or heating block. If the endotoxin concentration is expected in the range of 0.1 - 1 EU/ml, incubate at 37°C±1°C for T2. T1 Time ranges from 40 to 60 minutes, and T2 Time ranges from 8 to 16 minutes.
4.	After proper incubation, add 100 µl of reconstituted chromogenic substrate solution to each vial. Cap the vials and swirl gently to mix well. Do not shake or vortex to avoid foaming. Incubate at 37°C±1°C for 6 minutes.
5.	Add 500 µl of reconstituted Color-stabilizer #1 (Stop Solution) to each vial and swirl gently to mix well. Do not shake or vortex to avoid foaming. Add 500 µl of reconstituted Color-stabilizer #2 to each vial and mix well. Finally add 500 µl of reconstituted Color-stabilizer #3 to each vial. Gently swirl each vial to mix well. Bubbles must be avoided.
6.	Read the absorbance of each reaction vial at 545 nm using distilled water as blank to adjust the photometer to zero absorbance.
Waste disposal
Waste	Place to dispose	What to avoid
LAL vials	Place in a container with chlorine solution	N/A
Endotoxin residues	Sterilize.	N/A
Safety Notes
Every step must be done in the hood to avoid contamination Gloves must be worn at all times
References
ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit Genscript <a href="http://www.genscript.com/document/scm_files/prod"> http://www.genscript.com/document/scm_files/prod</a>

Edited by	Eduardo Serna Morales
Date	September 28th, 2013
Revision

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