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From 2013.igem.org

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Lab work

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Experiment 1: Preparation of growth mediums (MRS and LB) (June 5th, 2013)

10 Agar LB plates

15 Agar MRS plates

Notes:

• (1) 18.6g of Agar MRS were weighted to prepare 300ml of medium
• (2) 7g of Agar LB were weighted to prepare 200ml of medium
• (1) 5g of A-MRS were diluted in 80ml of distilled water
• Another 5g and 170ml of distilled water were added
• Small lumps were formed in the bottom of the Erlenmeyer flask
• (2) 7g of Agar LB were distilled in 100ml of distilled water
• Further 100ml of distilled water were added
• It wasn’t possible to use a control of the sterilization because there was no autoclave tape
• The autoclave began heating up
• The autoclave valve was lowered
• When the temperature of the autoclave reached 120⁰c, the power was cut to the half
• The vapor cycle ended and the temperature decreased
• The temperature reached 105⁰c and the valve was opened. The mediums were left inside the autoclave until de laminar flow cabinet was unoccupied
• The medium in petri dishes were stored in the refrigerator
• The growth mediums in storage were revised, they do not show signs of contamination

17 MRS Agar

9 LB Agar

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Experiment 2: Preparation of MRS broth and glycerol stocks (April 3rd, 2013)

• 0.7679g of powder for MRS broth were weighted
• 15ml of distilled water were measured in a 100ml measuring cylinder
• Besides, 200ml were prepared for future use
• 10.009g of powder for MRS broth were weighted
• It was diluted in 100ml of distilled water
• Another 100ml of distilled water were added
• 0.7679g of powder for MRS broth were diluted in 15ml of distilled water
• Both flasks were marked with autoclave tape and they were sterilized for 15 minutes at 121⁰c
• They cooled down and the autoclave tape confirmed the sterilization
• The 200ml broth was stored properly labeled
• The 15ml of MRS broth and the 15ml of glycerol were mixed to 100% in the laminar flow cabinet. They were gently shaken until an homogenous mixture was obtained in a falcon tube of 45ml
• An aliquot of L. Plantarum was added to the broth and glycerol solution and it was sealed with parafilm
• It was left incubating at 37⁰c and 200rpm
• After 24 hours the sample was taken out of the incubator to striate it
• 2500 (x2)ml were placed in 22 eppendorf tubes using a micropipette of 200µl to cultivate and recompile the mixture of L. Plantarum contained in the falcon tube
• 1 eppendorf tube was centrifuged at 13.4 rpm for 3 minutes
• After the centrifugation there was a small pellet left, from which the supernatant was discarded and a solution of MRS broth (500ml) was added
• Once the mixture was made, two petri dishes with MRS Agar were striated in the laminar flow cabinet. A third dish was striated fin case of an emergency
• The petri dishes were placed facing down in the incubator at 37⁰c and 200 rpm
• The remaining samples (21 falcon tubes) were stored in deep freezing
• E. Coli K12 was striated in two petri dishes with LB Agar
• They were placed in the incubator at 37⁰c.