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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)