Team:BIOSINT Mexico/Protocols

From 2013.igem.org

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'''Protocol 1: 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
 +
 +
 +
----
 +
'''Protocol 2: 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.
 +
 +
 +
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 +
'''Protocol 3: 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)

Revision as of 01:02, 28 September 2013

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Protocols



Protocol 1: 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



Protocol 2: 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.



Protocol 3: 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)