Team:BIOSINT Mexico/Protocols

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<html><h1 class="myowntitle">Protocols</h1> </html>
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'''Preparation of growth mediums (MRS and LB)'''
 +
 +
*Weight 18.6 g of Agar MRS to prepare 300ml of medium
 +
*Weight 7 g of Agar LB to prepare 200ml of medium
 +
*Dilute 5 g of A-MRS in 80 ml of distilled water
 +
*Add 5 g and 170 ml of distilled water
 +
*Dilute 7 g of Agar LB in 100ml of distilled water
 +
*Add 100ml of distilled water
 +
*Place the autoclave tape
 +
*Set the autoclave
 +
*When the temperature of the autoclave reaches 120⁰c, cut the power by half
 +
*When the temperature reaches 105⁰c, open the valve.
 +
*Retrieve the mediums
 +
 +
 +
----
 +
'''Preparation of MRS broth and glycerol stocks'''
 +
 +
*Weight 0.7679 g of powder for MRS broth
 +
*Measure 15ml of distilled water in a 100ml measuring cylinder
 +
*Weight 10.009g of powder for MRS broth
 +
*Dilute it in 100ml of distilled water
 +
*Add 100ml of distilled water
 +
*Dilute 0.7679g of powder for MRS broth in 15ml of distilled water
 +
*Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c
 +
*Label the broth
 +
*Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml
 +
*Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm
 +
*Incubate it at 37⁰c and 200rpm
 +
*After 24 hours, take the sample out of the incubator to striate it
 +
*Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube
 +
*Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes
 +
*Discard the supernatant and add a solution of MRS broth (500ml)
 +
*Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet
 +
*Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm
 +
*Store the remaining samples (21 falcon tubes) in deep freezing
 +
*Striate E. Coli K12 in two petri dishes with LB Agar
 +
*Place the dishes in the incubator at 37⁰c.
 +
 +
 +
----
 +
'''Plasmid purification'''
 +
 +
*Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times
 +
*Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth
 +
*Centrifuge the eppendorf tube for 15min at 6000 rpm
 +
*Discard the supernatant
 +
*Resuspend the pellet in 250ml resuspension buffer
 +
*Add 250ml of Lysis Buffer L7
 +
*Gently mix the tube carefully inverting it 5 times carefully
 +
*Add and mix softly 350ml of precipitation Buffer N4, inverting the tube
 +
*Centrifuge it at 12000 rpm for 10 minutes
 +
*Transfer the supernatant into a spin column inside a washtube
 +
*Centrifuge it at 12000 rpm for a minute
 +
*Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column
 +
*Incubate it for one minute at room temperature
 +
*Centrifuge the column at 12000 rpm for 1 minute
 +
*Discard the liquid from the washtube and place the column inside the tube
 +
*Add 700ml of Wash Buffer W9 with ethanol to the column
 +
*Centrifuge the column with the washtube at 12000 rpm for 1 minute
 +
*Discard the liquid from the washtube
 +
*Centrifuge the column with the washtube at 12000 rpm for 1 minute
 +
*Discard the liquid from the washtube
 +
*Place the column inside an eppendorf tube of 1.5ml
 +
*Add 75ml of preheated TE Buffer at the center of the column
 +
*(Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes)
 +
*Incubate the column for 1 minute at room temperature
 +
*The column was centrifuged at 12000 rpm for 2 minutes
 +
*(The eppendorf tube contains the purified plasmid)
 +
 +
 +
----
 +
'''Preparation of TE buffer'''
 +
 +
*Dilute 6.05 g of TRIS in 60 ml of distilled water
 +
*Dilute 9.3041 g of EDTA in 60 ml of distilled water
 +
*Add HCl until the TRIS’ pH reaches 8.3
 +
*Add NaOH crystals until the EDTA’S pH reaches 7.79
 +
*Seal and autoclave for 15 minutes at 121⁰c
 +
 +
 +
----
 +
'''Preparation of competent E. Coli cells'''
 +
 +
*Prepare a falcon tube with 50 ml of CaCl2 0.1M
 +
*Prepare another falcon tube with CaCl2 0.1M/ 15% glycerol
 +
*Fill 4 eppendorf tubes of 1.5 ml with the E. Coli culture
 +
**2 of them with 1.5 ml each of E. Coli culture in LB broth (1)
 +
**2 of them with 1.5 ml each of E. Coli culture in LB broth (2)
 +
*Leave the 4 tubes in ice for 10 minutes
 +
*Centrifuge the 4 tubes for 3 minutes at 6000 rpm and discard the supernatant
 +
*Add 1.5 ml of the culture to each tube
 +
*Centrifuge the tubes for 3 minutes at 6000 rpm
 +
*Discard the supernatant
 +
*gently resuspend the pellet with 1.2 ml CaCl2 0.1M for each tube
 +
*Incubate the 4 tubes in ice for 20 minutes
 +
*Centrifuge the tubes for 3 minutes at 6000 rpm
 +
*Discard the supernatant
 +
*Resuspend the pellets with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
 +
*Pour them into micro-tubes (300 µl/tube), seal them and freeze them at -80⁰c
 +
 +
 +
----
 +
'''Electrophoresis gel for L. Plantarum'''
 +
 +
Agarose preparation
 +
*Prepare TAE 1x with 392 ml of distilled water and 8 ml of TAE 50x (for a 400 ml solution)
 +
*Add 0.35 g to 35 ml (grams of agarose)
 +
Preparation of the sample to be placed in the gel
 +
*Place 10 µl of each sample of purified plasmid in eppendorf tubes of 1.5 ml
 +
*Add 5 µl of 6x DNA Loading Dye to each one, pipetting until completely homogenizing the sample
 +
Running the gel
 +
*Pour the agarose mixture into the casting tray for 35 ml gels with their respective well combs
 +
*Remove the well combs once it has solidified
 +
*Place the gel in the gel box, with the wells on the side of the anode
 +
*Fill the box with TAE 1x up to the indicated measure (equal quantities on both sides of the box)
 +
*Pour 15 µl of gene ruler in the first well with a micro pipet
 +
*Pour 15 µl of each sample in consecutive wells with a micro pipet
 +
*Close the electrophoresis box and connect the power line
 +
*Set the current to 100 V and leave it running for 55 minutes
 +
*Turn off the current and remove the gel from the chamber
 +
*Leave the gel rocking in Ethidium bromide for 15 minutes (always use gloves when handling Ethidium bromide)
 +
*Take the gel out of the platform and of the Ethidium bromide and place it in the transiluminator
 +
*Observe the results with UV light
 +
 +
 +
----
 +
'''Rehydration of plasmids in kit plates'''
 +
 +
*Number the plate kits as indicated in the iGEM’s parts registry and the plasmids to be used
 +
*Pierce the protective layer with a micro-pipet
 +
*Rehydrate each plasmid with 10 µl of distilled water
 +
*Pipet the liquid until it is homogeneous
 +
*It was left resting for 5 minutes to ensure its rehydration
 +
 +
 +
----
 +
'''Preparation of calcium chloride buffer for transformation 200mM'''
 +
 +
*Sterilize distilled water in the autoclave for 15 minutes at 121⁰c
 +
*Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water
 +
*Fill a syringe to its maximum capacity with the buffer
 +
*Connect a ministart 0.2µm filter to the syringe
 +
*Pour the content of the syringe into a sterile falcon tube through the filter
 +
*Seal it and place it in refrigeration
 +
 +
 +
----
 +
'''Preparation of transformation buffer CaCl2 50mM'''
 +
 +
*Place 1 ml of CaCl2 buffer 200mM in a beaker
 +
*Add 3 ml of sterile distilled water were added
 +
*Fill a syringe to its maximum capacity with the buffer
 +
*Connect A ministart 0.2µm to the syringe
 +
*Pour the content of the syringe into a sterile falcon tube through the filter
 +
*Refrigerate
 +
 +
 +
----
 +
'''E. Coli transformation with pGLO (GFP)'''
 +
 +
*Take the competent cells out of deep freeze and let them defrost in ice
 +
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube
 +
*Add 10 µl of pGLO
 +
* Gently mix them and leave them incubating in ice for 30 minutes
 +
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds
 +
*Cool the tube in ice for 5 minutes
 +
*Add 900 µl of S.O.C. to the tube
 +
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm
 +
*Striate a control dish and a dish with antibiotic
 +
*Seal both dishes and leave them incubating facing down at 37⁰c
 +
 +
 +
----
 +
'''Preparation of plates with LB agar and antibiotic'''
 +
 +
*Heat the LB agar was in the electrical stove until it is completely liquefied
 +
*The next plates were prepared:
 +
Plate with kanamycin
 +
*Pour 40 µl of kanamycin and 40 ml of LB agar into a beaker (for 2 dishes)
 +
*Mix them and pour them into 2 dishes
 +
Plate with kanamycin and L-arabinose
 +
*Mix 20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar in a beaker
 +
*Pour the solution into a petri dish
 +
Plate with ampicillin
 +
*Mix 20 µl of ampicillin and 20 ml of agar in a beaker
 +
*Pour the solution into a dish
 +
*Let the plates solidify and label them
 +
 +
----
 +
'''E. Coli transformation with parts promoter 1,2 and rbs '''
 +
 +
*Take the competent cells out of deep freeze and let them defrost in ice
 +
*Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube
 +
*Add 10 µl of pGLO
 +
* Gently mix them and leave them incubating in ice for 30 minutes
 +
*Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds
 +
*Cool the tube in ice for 5 minutes
 +
*Add 900 µl of S.O.C. to the tube
 +
*Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm
 +
*Striate a control dish and a dish with antibiotic
 +
*Seal both dishes and leave them incubating facing down at 37⁰c

Latest revision as of 04:02, 28 September 2013

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Protocols

Preparation of growth mediums (MRS and LB)

  • Weight 18.6 g of Agar MRS to prepare 300ml of medium
  • Weight 7 g of Agar LB to prepare 200ml of medium
  • Dilute 5 g of A-MRS in 80 ml of distilled water
  • Add 5 g and 170 ml of distilled water
  • Dilute 7 g of Agar LB in 100ml of distilled water
  • Add 100ml of distilled water
  • Place the autoclave tape
  • Set the autoclave
  • When the temperature of the autoclave reaches 120⁰c, cut the power by half
  • When the temperature reaches 105⁰c, open the valve.
  • Retrieve the mediums


Preparation of MRS broth and glycerol stocks

  • Weight 0.7679 g of powder for MRS broth
  • Measure 15ml of distilled water in a 100ml measuring cylinder
  • Weight 10.009g of powder for MRS broth
  • Dilute it in 100ml of distilled water
  • Add 100ml of distilled water
  • Dilute 0.7679g of powder for MRS broth in 15ml of distilled water
  • Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c
  • Label the broth
  • Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml
  • Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm
  • Incubate it at 37⁰c and 200rpm
  • After 24 hours, take the sample out of the incubator to striate it
  • Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube
  • Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes
  • Discard the supernatant and add a solution of MRS broth (500ml)
  • Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet
  • Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm
  • Store the remaining samples (21 falcon tubes) in deep freezing
  • Striate E. Coli K12 in two petri dishes with LB Agar
  • Place the dishes in the incubator at 37⁰c.


Plasmid purification

  • Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times
  • Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth
  • Centrifuge the eppendorf tube for 15min at 6000 rpm
  • Discard the supernatant
  • Resuspend the pellet in 250ml resuspension buffer
  • Add 250ml of Lysis Buffer L7
  • Gently mix the tube carefully inverting it 5 times carefully
  • Add and mix softly 350ml of precipitation Buffer N4, inverting the tube
  • Centrifuge it at 12000 rpm for 10 minutes
  • Transfer the supernatant into a spin column inside a washtube
  • Centrifuge it at 12000 rpm for a minute
  • Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column
  • Incubate it for one minute at room temperature
  • Centrifuge the column at 12000 rpm for 1 minute
  • Discard the liquid from the washtube and place the column inside the tube
  • Add 700ml of Wash Buffer W9 with ethanol to the column
  • Centrifuge the column with the washtube at 12000 rpm for 1 minute
  • Discard the liquid from the washtube
  • Centrifuge the column with the washtube at 12000 rpm for 1 minute
  • Discard the liquid from the washtube
  • Place the column inside an eppendorf tube of 1.5ml
  • Add 75ml of preheated TE Buffer at the center of the column
  • (Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes)
  • Incubate the column for 1 minute at room temperature
  • The column was centrifuged at 12000 rpm for 2 minutes
  • (The eppendorf tube contains the purified plasmid)


Preparation of TE buffer

  • Dilute 6.05 g of TRIS in 60 ml of distilled water
  • Dilute 9.3041 g of EDTA in 60 ml of distilled water
  • Add HCl until the TRIS’ pH reaches 8.3
  • Add NaOH crystals until the EDTA’S pH reaches 7.79
  • Seal and autoclave for 15 minutes at 121⁰c


Preparation of competent E. Coli cells

  • Prepare a falcon tube with 50 ml of CaCl2 0.1M
  • Prepare another falcon tube with CaCl2 0.1M/ 15% glycerol
  • Fill 4 eppendorf tubes of 1.5 ml with the E. Coli culture
    • 2 of them with 1.5 ml each of E. Coli culture in LB broth (1)
    • 2 of them with 1.5 ml each of E. Coli culture in LB broth (2)
  • Leave the 4 tubes in ice for 10 minutes
  • Centrifuge the 4 tubes for 3 minutes at 6000 rpm and discard the supernatant
  • Add 1.5 ml of the culture to each tube
  • Centrifuge the tubes for 3 minutes at 6000 rpm
  • Discard the supernatant
  • gently resuspend the pellet with 1.2 ml CaCl2 0.1M for each tube
  • Incubate the 4 tubes in ice for 20 minutes
  • Centrifuge the tubes for 3 minutes at 6000 rpm
  • Discard the supernatant
  • Resuspend the pellets with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
  • Pour them into micro-tubes (300 µl/tube), seal them and freeze them at -80⁰c


Electrophoresis gel for L. Plantarum

Agarose preparation

  • Prepare TAE 1x with 392 ml of distilled water and 8 ml of TAE 50x (for a 400 ml solution)
  • Add 0.35 g to 35 ml (grams of agarose)

Preparation of the sample to be placed in the gel

  • Place 10 µl of each sample of purified plasmid in eppendorf tubes of 1.5 ml
  • Add 5 µl of 6x DNA Loading Dye to each one, pipetting until completely homogenizing the sample

Running the gel

  • Pour the agarose mixture into the casting tray for 35 ml gels with their respective well combs
  • Remove the well combs once it has solidified
  • Place the gel in the gel box, with the wells on the side of the anode
  • Fill the box with TAE 1x up to the indicated measure (equal quantities on both sides of the box)
  • Pour 15 µl of gene ruler in the first well with a micro pipet
  • Pour 15 µl of each sample in consecutive wells with a micro pipet
  • Close the electrophoresis box and connect the power line
  • Set the current to 100 V and leave it running for 55 minutes
  • Turn off the current and remove the gel from the chamber
  • Leave the gel rocking in Ethidium bromide for 15 minutes (always use gloves when handling Ethidium bromide)
  • Take the gel out of the platform and of the Ethidium bromide and place it in the transiluminator
  • Observe the results with UV light


Rehydration of plasmids in kit plates

  • Number the plate kits as indicated in the iGEM’s parts registry and the plasmids to be used
  • Pierce the protective layer with a micro-pipet
  • Rehydrate each plasmid with 10 µl of distilled water
  • Pipet the liquid until it is homogeneous
  • It was left resting for 5 minutes to ensure its rehydration


Preparation of calcium chloride buffer for transformation 200mM

  • Sterilize distilled water in the autoclave for 15 minutes at 121⁰c
  • Dilute 1.1159 g of calcium chloride in 50 ml of sterile distilled water
  • Fill a syringe to its maximum capacity with the buffer
  • Connect a ministart 0.2µm filter to the syringe
  • Pour the content of the syringe into a sterile falcon tube through the filter
  • Seal it and place it in refrigeration


Preparation of transformation buffer CaCl2 50mM

  • Place 1 ml of CaCl2 buffer 200mM in a beaker
  • Add 3 ml of sterile distilled water were added
  • Fill a syringe to its maximum capacity with the buffer
  • Connect A ministart 0.2µm to the syringe
  • Pour the content of the syringe into a sterile falcon tube through the filter
  • Refrigerate


E. Coli transformation with pGLO (GFP)

  • Take the competent cells out of deep freeze and let them defrost in ice
  • Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube
  • Add 10 µl of pGLO
  • Gently mix them and leave them incubating in ice for 30 minutes
  • Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds
  • Cool the tube in ice for 5 minutes
  • Add 900 µl of S.O.C. to the tube
  • Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm
  • Striate a control dish and a dish with antibiotic
  • Seal both dishes and leave them incubating facing down at 37⁰c


Preparation of plates with LB agar and antibiotic

  • Heat the LB agar was in the electrical stove until it is completely liquefied
  • The next plates were prepared:

Plate with kanamycin

  • Pour 40 µl of kanamycin and 40 ml of LB agar into a beaker (for 2 dishes)
  • Mix them and pour them into 2 dishes

Plate with kanamycin and L-arabinose

  • Mix 20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar in a beaker
  • Pour the solution into a petri dish

Plate with ampicillin

  • Mix 20 µl of ampicillin and 20 ml of agar in a beaker
  • Pour the solution into a dish
  • Let the plates solidify and label them

E. Coli transformation with parts promoter 1,2 and rbs

  • Take the competent cells out of deep freeze and let them defrost in ice
  • Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube
  • Add 10 µl of pGLO
  • Gently mix them and leave them incubating in ice for 30 minutes
  • Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds
  • Cool the tube in ice for 5 minutes
  • Add 900 µl of S.O.C. to the tube
  • Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm
  • Striate a control dish and a dish with antibiotic
  • Seal both dishes and leave them incubating facing down at 37⁰c
Retrieved from "http://2013.igem.org/Team:BIOSINT_Mexico/Protocols"