Team:Tec-Monterrey/ITESM14 protocols.html

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

Protocol 1-Calcium competent cells

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

Protocol 2-Transformation of Calcium Competent Cells

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

Protocol 3-Miniprep

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
Qiagen. (2009, November 20). Miniprep/Qiagen kit protocol. Retrieved June 2, 2014, from http://openwetware.org/wiki/Miniprep/Qiagen_kit_protocol
Edited by	Mercedes Vázquez
Date	July 1st, 2013
Revision	September 10th, 2014

Protocol 4-Electro-competent cells

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

Protocol 5-Transformation of electro-competent cells

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

Protocol 6-Digestion with restriction enzymes

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.
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 https://www.neb.com/protocols/2014/05/07/double-digest-protocol-with-standard-restriction-enzymes
Optimizing Restriction Endonuclease Reactions https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
Digestion Protocol for BioBrick Assembly Kit (E0546) https://www.neb.com/protocols/1/01/01/digestion-protocol-e0546
Edited by	Eduardo A. Ramírez Rodríguez
Date	September 9th, 2014
Revision

Protocol 7-Agarose based electrophoresis

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 Acetate (100% acetic acid): 57.1 ml EDTA: 100 ml 0.5M sodium EDTA Add dH2O up to one litre.
Related protocols
Digestion, enzyme restriction analysis.
Steps
Prepare the gel
1.	The amount of agarose depends on the size of the DNA. (A guide can be seen in the table below)
	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
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.
3.	Microwave until the agarose is fully melted and cool down a few minutes.
4.	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.
Load the gel
5.	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).
6.	Pour the mixture in the wells. Be careful with the bubbles and breaking the gel with the pipette.
7.	Fill the chamber with TAE and run the gel 70 min at 90 V and ~180 mA
Stain and visualize the gel
8.	Stain the gel soaking it in EtBr, be careful not to break it.
9.	Reveal usin the transiluminator, first focus using the white light lamp and use the UV to make the bands visible.
Waste disposal
Waste Place to dispose What to avoid TAE Throw through the sink, the school has a water treatment plant for all the facilities. N/A Gel 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 Do not touch the gel or the EtBr solution with your bare hands EtBr solution Every once in a while the solution must be changed, and the discarded solution must be See above Gloves You must discard the glove you use when you handle the gel and the computer. They must be thrown in the EtBr labeled container. Make sure to dispose your gloves
Safety Notes
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.
References
Agarose Gel Electrophoresis http://www.addgene.org/plasmid-protocols/gel-electrophoresis/
Edited by	Eduardo A. Ramírez Rodríguez
Date	September 28th, 2014
Revision

Protocol 8-Cell Lysis and Protein Purification

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:
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 HisPurTM Ni-NTA Purification Kit https://www.piercenet.com/instructions/2162206.pdf
Edited by	Eduardo Serna Morales
Date	September 28th, 2014
Revision

Protocol 9-Red Lambda Protocol

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
Digestion. Electrocompetent cells/ Calcium competent cells.
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
Murphy et al. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol. 2003 Dec 13. 4():11.
Edited by	Mercedes Vázquez
Date	September 25th, 2014
Revision

Protocol 10-Quorum Sensing Assay

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).
6.	Measure fluorescence of GFP (395nm excitation, 509nm emission, 495nm cutoff).
7.	Measure cell density at 600nm.
8.	Repeat steps 3 to 7. (For better characterization it is recommended to repeat this steps for 16-20 hours)
Waste disposal
Waste Place to dispose What to avoid LB medium Place in a container with chlorine solution N/A Microplate 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	Mercedes Vázquez
Date	September 25th,2014
Revision

Protocol 11-Phage Purification by Isoelectric Precipitation

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. Culture conditions: 37°C 220 rpm 16 h
Bacterial cells are removed by centrifugation (5,000 rpm) for 10 min and a second centrifugation (12,000 rpm) for 10 min.
Steps
1.	Adjust he pH of the supernatant to pH 4.2 with 5M HCl and is mixed briefly.
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.
3.	Remove the residual LB, suspend pellets in 40mL of Mili-Q water by vortexing and centrifuge (13,000 rpm) for 10min at 20°C.
4.	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.
5.	Adjust the respuspended pellets pH to 7 with 5M NaOH and keep samples at 4°C.
Waste disposal
Waste Place to dispose What to avoid LB medium Place in a 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
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
Edited by	Mercedes Vázquez
Date	September 25th, 2014
Revision

Protocol 12-Phage Quantification by Spectrophotometry

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:
	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 Phage 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.
Edited by	Mercedes Vázquez
Date	September 25th, 2014
Revision

Protocol 13-Tricine-SDS-PAGE

Objective
Separate proteins according to their electrophoretic mobility.
Material
Glycerol (Serva)
Tetramethylethylenediamine (TEMED;Sigma)
Mercaptoethanol (Sigma)
Ammonium persulfate (Sigma)
Urea (Serva)
Reducing sample buffers: Buffer A: 12% SDS (wt/vol), 6% mercaptoethanol (vol/vol), 30% glycerol(wt/vol), 0.05% Coomassie blue G-250 (Serva), 150 mM Tris/HCl (pH 7.0) Buffer A/4: buffer A diluted with 3 volumes of water Buffer C: buffer A without glycerol
Nonreducing sample buffers: Buffer B: 12% SDS (wt/vol), 30% glycerol (wt/vol), 0.05% Coomassie blue G-250 (Serva), 150 mM Tris/HCl (pH 7.0) Buffer B/4: buffer B diluted with 3 volumes of water Buffer D: buffer B without glycerol
Equipment & Apparatus
Vertical electrophoresis apparatus (Bio Rad)
Power Supply (600V, 500mA) for SDS-PAGE and electroblotting
Polyvinylidene fluoride (PVDF) membranes or nitrocellulose membranes
Previous steps
Prepare Reducing and nonreducing sample buffers.
Prepare the Electrode and gel buffers for Tricine–SDS-PAGE as follows:
Anode Buffer(10x) Cathode Buffer(10x) Gel Buffer(3x) Tris (M) 1.0 1.0 3.0 Tricine (M) - 1.0 - HCl (M) 0.225 - 1.0 SDS (%) - 1.0 0.3 pH 8.9 -8.25 8.45
Steps
1.	Select and cast the appropriate base (separating) gel. The amounts of reagents required for two gels with dimensions 0.07 × 14 × 14 cm and containing 10% and 16% acrylamide are given in the table that follows. Do not degas the gel mixtures, because the gel buffer contains SDS. Overlay the poured gels with several drops of water. Leave the gels for about 30 min to polymerize. The freshly prepared ammonium persulfate (APS) solution and TEMED should be added last, immediately before pouring the gels, because these polymerize the gels.
	4% Sample Gel 10% gel 16% gel 16%/6 M urea AB-3 (ml) 1 6 10 10 Gel Buffer (3x) 3 10 10 10 Glycerol (g) - 3 3 - Urea (g) - - - 10.8 Add water to final volume (ml) 12 30 30 30 Polymerize by adding: APS (10%) (µl) 90 150 100 100 TEMED (µl) 9 15 10 10
2.	Overlay the polymerized separating gel (10% or 16%, or 16%/6 M urea) directly with a 4% sample (stacking) gel, except if resolution of proteins <5 kDa is desired. If resolution of proteins <5 kDa is desired, then use AB-6 instead of AB-3 for the separating gel and overlay the separating gel with a 1-cm 10% gel. The 16% separating gel and the overlaid 10% ‘spacer gel’ can be polymerized together if no glycerol is added to the 10% acrylamide gel mixture (the common role of glycerol in SDS gels is to increase the density of solutions and to facilitate gel casting; it has no obvious effect on protein separation). Introducing a 10% ‘spacer gel’ between 4% stacking and 16% separating gels considerably sharpens the bands for proteins and peptides of 1–5 kDa.
3.	Adjust protein concentrations so that a suitable amount of protein can be loaded onto the gel. Concentrate samples, preferentially by techniques that do not increase the salt concentration (such as ultrafiltration). Roughly 0.2–1 µg of protein for each protein band (in 0.7 × 5 mm gel strips) is sufficient for Coomassie staining. Accordingly, the desired protein concentration in the sample is 0.1 mg ml–1 for each protein band.
4.	Mix samples with SDS-containing sample buffers. The volume and buffer to be used depend on the origins of the samples. For low-density samples such as elution fractions from chromatographic columns, add 5 µl of reducing or nonreducing sample incubation buffer A or B to 15 µl of sample. For high-density samples such as fractions from sucrose density gradients, add 5 µl of sample buffer C or D to 15 µl of sample. For pellet samples, resuspend the pellet in 15–20 µl of buffer A/4 or B/4.
5.	Incubate samples at 37 °C for 15 min or for up to 60 min for samples that were in pellet form. Avoid boiling samples, because membrane proteins can irreversibly aggregate in SDS at temperatures >50 °C.
6.	Mount the gels in the vertical electrophoresis apparatus, and add anode buffer as the lower electrode buffer and cathode buffer as the upper electrode buffer.
7.	Load samples under the cathode buffer. Apply 10-µl sample volumes to 0.7 × 5 mm sample wells.
8.	Set running conditions appropriate to your type of gel; guidance on appropriate running conditions are given in the table below.
	10% (0.7mm) 16% (0.7mm) 16% (1.6mm) Initial voltage 30V 30V 30V Next voltage step 190V 200V 90V constant Voltage at end of run 270V 300V - Time 3-4h 5-6h Overnight
9.	Protein can be visualized directly in the gel by Coomassie staining or silver staining. Both Coomassie staining and silver staining can be done providing Coomassie staining is done first. Both procedures are compatible with subsequent mass spectrometric analysis. Alternatively, the proteins can be transferred to a PVDF membrane by electroblotting.
Waste disposal
Waste Place to dispose What to avoid Liquid and gel material Discarded through hazardous waste management system. Avoid having direct contact with it.
Safety Notes
Gloves must be worn at all times. All the procedure must be done in a restricted area.
References
Schägger, H. (2006). tricine–sds-page. Nature protocols, 1(1), 16-22.
Edited by	Mercedes Vázquez
Date	October 15th, 2014
Revision

Protocol 14-Chromogenic Lal Endotoxin Assay

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 http://www.genscript.com/document/scm_files/prod
Edited by	Eduardo Serna Morales
Date	September 28th, 2013
Revision

Protocol 15-Polymerase Chain Reaction (PCR)

Objective
DNA amplification using Taq DNA polymerase.
Material
Nuclease free water
dNTPs
10X ThermoPol or Standard Taq Reaction Buffer
Taq DNA Polymerase
Primers
Equipment & Apparatus
Thermocycler
Laminar flow hood (Scientific Visions)
PCR Tubes
Microcentrifuge (Thermo)
Previous steps
Design Primers. Serves as a starting point for DNA synthesis. Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%.
Steps
1.	Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice:
	Concentration 25µL reaction 50 µL reaction Final Concentration 10X ThermoPol or Standard Taq Reaction Buffer 2.5 µL 5 µL 1X 10 mM dNTPs 0.5 µL 1 µL 200 µM 10 µM Forward Primer 0.5 µL 1 µL 0.2 µM(0.05-1mM) 10 µM Reverse Primer 0.5 µL 1 µL 0.2 µM(0.05-1mM) Template DNA Variable Variable <1000ng Taq DNA Polymerase 0.125 µL 0.25 µL 1.25units/50 µL PCR Nuclease-free water To 25 µL To 50 µL
	Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
2.	Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling.
3.	Thermocycling conditions for a routine PCR:
	Step Temperature Time Initial Denaturation 95°C 30sec 30 cycles 95°C 45-68°C 68°C 15-30sec 15-60sec (30 cycles) 1min/kb Final Extension 72°C 5 minutes Hold 4-10°C
Waste disposal
Waste Place to dispose What to avoid Waste Place in container with chlorine solution. N/A
Safety Notes
Gloves must be worn at all times. Must be done in clean areas to avoid contamination.
References
NEB PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273) https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273
Edited by	Eduardo Serna Morales
Date	September 28th, 2014
Revision

Protocol 16-Apoptosis Detection

Objective
Detect apoptosis in mammalian cells through the use of FITC Annexin V and 7-AAD
Approximate time
60 min
Material
FITC Annexin V (Biolegend)
7-AAD Viability Staining Solution (Biolegend)
Annexin V Binding Buffer (Biolegend)
Cell Staining Buffer (Biolegend)
Equipment & Apparatus
Flow cytometer
Laminar flow hood (Scientific Visions)
Previous steps
The treatment with the effectors should be done during different time lapses before the apoptosis staining.
Steps
1.	Wash cells twice with cold BioLegend's Cell StainingBuffer, and then resuspend cells in Annexin V Binding Buffer at a concentration of 0.25-1.0 x 107 cells/ml.
2.	Transfer 100 µl of cell suspension in a 5 ml test tube.
3.	Add 5 µl of FITC Annexin V.
4.	Add 5 µl of 7-AAD Viability Staining Solution.
5.	Gently vortex the cells and incubate for 15 min at room temperature (25°C) in the dark.
6.	Add 400 µl of Annexin V Binding Buffer to each tube. Analyze by flow cytometry with proper machine settings.
Waste disposal
Waste Place to dispose What to avoid Cell Staining Buffer Collect in a flask and dispose with the help of a specialized company N/A Cytometry Solution with cells Collect in a flask and dispose with the help of a specialized company Avoid contact with 7-AAD
Safety Notes
7-AAD is a potential carcinogen. It is recommended that the user wear protective clothing, gloves, and eye/face protection in order to avoid contact with skin and eyes.
References
FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend) http://www.biolegend.com/fitc-annexin-v-apoptosis-detection-kit-with-7-aad-8264.html
Edited by	Claudia Nallely Alonso Cantú
Date	October 9th, 2014
Revision

Protocol 17-Western Blot

Objective
To detect specific proteins in a sample.
Material
Antibody Diluent
10X Wash Buffer
HRP Reagent
Detection Reagent 1
Primary Antibody
Equipment & Apparatus
Rotary platform shaker for agitation of membrane during incubations
Blot Gel Tank (Bio-rad)
Power Supply
Membrane to transfer the protein
Previous steps
Prepare reagents:
Reaction volume Restriction Enzyme Fast Western 1X Wash Buffer Mix 1 part of the Fast Western 10X Wash Buffer with 9 parts of water. Primary Antibody Working Solution Shake the Fast Western Antibody Diluent well before use. Dilute the primary antibody with diluent to a concentration ranging from 0.2 to10 μg/ml. Use ~0.125 ml of antibody per cm2 of membrane (e.g., 10 ml per 8 × 10 cm blot). The stability of diluted primary antibodies varies depending on the antibody. For best results prepare working dilution immediately before use. Fast Western Optimized HRP Reagent Working Dilution Mix 1 part of Fast Western Optimized HRP Reagent with 9 parts of Fast Western Antibody Diluent. Use 0.125 ml per cm2 of membrane (e.g., 10 ml per 8 × 10 cm blot). For best results, use this solution within 1 hour. Detection Reagent Working Solution Mix Detection Reagents 1 and 2 at 1:1. Use 0.125 ml Working Solution per cm2 of membrane (e.g., 10 ml per 8 × 10 cm blot). For best results prepare working solution immediately before use (Step 9). The working solution is stable for up to 1 hour at room temperature.
Transfer of proteins: Detailed instructions for the transfer process can be found on the websites of the manufacturers of transfer apparatus, and will vary depending on the system. The principle is the same in each case though. In wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane. The sandwich is submerged in transfer buffer to which an electrical field is applied. The negatively-charged proteins travel towards the positively-charged electrode, but the membrane stops them, binds them, and prevents them from continuing on. A standard buffer for wet transfer is the same as the 1X Tris-glycine buffer used for the migration/running buffer without SDS but with the addition of methanol to a final concentration of 20%. For proteins larger than 80 kD, it is recommended that SDS is included at a final concentration of 0.1%.
Steps
1.	Remove blot from the transfer apparatus and place in a clean incubation tray.
2.	Briefly wash blot in Fast Western 1X Wash Buffer to remove transfer buffer.
3.	Add the Primary Antibody Working Dilution to the blot and incubate for 30 minutes at room temperature (RT) with shaking.
4.	Remove blot from the primary antibody solution and place it in a clean incubation tray.
5.	Add the Fast Western Optimized HRP Reagent Working Dilution and incubate for 10 minutes at RT with shaking. Higher sensitivity can be obtained by increasing the incubation time to 15 minutes; however, one or more additional washes will be required to reduce background.
6.	Remove blot from the HRP solution and place it in a clean incubation tray.
7.	Wash membrane by suspending it in approximately 20 ml of Fast Western 1X Wash Buffer and agitating for 5 minutes. Repeat this wash twice.
8.	Remove blot and place it in a clean incubation tray. Add the Detection Reagent Working Solution and incubate for 1-5 minutes at RT.
9.	Remove blot from Detection Reagent Working Solution and place it in a plastic sheet protector or clear plastic wrap. Use an absorbent tissue to remove excess liquid and to carefully press out any bubbles from between the blot and the membrane protector.
10.	Expose the blot to film or use your preferred imaging method.
Waste disposal
Waste Place to dispose What to avoid Unpolymerized solutions Has to be disposed as hazardous waste. Avoid any contact Polimerized Gels Can be disposed in the trash. N/A
Safety Notes
Acrylamide is a neurotoxin. Gloves and face mask must be worn at all times. Wash hands thoroughly after use.
References
Pierce Fast Western Blot Kit https://www.piercenet.com/instructions/2162133.pdf
Definitive guide to Western Blot http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf
Edited by	Mercedes Vázquez
Date	October 15th, 2014
Revision