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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 (July 8th, 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.

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Experiment 3: Plasmid purification (July 16th, 2013)

• An eppendorf tube of 1.5ml with L. Plantarum culture was centrifuged at 12000 rpm for 1 minute two times
• The supernatant was discarded and the tube’s content was resuspended with more L. Plantarum culture in MRS broth
• The eppendorf tube was centrifuged for 15min at 6000 rpm
• The supernatant was discarded
• The pellet was resuspended in 250ml resuspension buffer
• 250ml of Lysis Buffer L7
• The tube was gently mixed inverting it 5 times carefully
• 350ml of precipitation Buffer N4 were added and mixed softly inverting the tube
• It was centrifuged at 12000 rpm for 10 minutes
• The supernatant was transferred to a spin column inside a washtube
• It was centrifuged at 12000 rpm for a minute
• The supernatant was discarded and 500 ml of Wash Buffer with ethanol (w10) were added to the column
• It was incubated for one minute at room temperature
• The column was centrifuged at 12000 rpm for 1 minute
• The liquid from the washtube was discarded and the column was placed inside the tube
• 700ml of Wash Buffer W9 with ethanol were added to the column
• The column with the washtube was centrifuged at 12000 rpm for 1 minute
• The liquid from the washtube was discarded
• The column in the washtube was centrifuged at 12000 rpm for 1 minute
• The liquid from the washtube was discarded
• The column was placed inside an eppendorf tube of 1.5ml
• 75ml of preheated TE Buffer were added at the center of the column
• The TE Buffer was previously warmed in water bath at 65⁰c-70⁰c for 3 minutes
• The column was incubated for 1 minute at room temperature
• The column was centrifuged at 12000 rpm for 2 minutes
• The eppendorf tube contains the purified plasmid

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Experiment 4: Preparation of mediums for lactobacillus and E. Coli (July 17th, 2013)

• 15.3 g of MRS were weighted for 300 ml, the 2% (0.306 g.) of the MRS was used to do agar-agar
• The autoclave started to heat
• 8.75 g of LB agar were weighted to dilute in 500 ml of distilled water
• 0.5005 g of LB broth were weighted to be diluted in 250 ml of distilled water
• The 3 mediums were introduced in the autoclave
• After 54 minutes the sterilization was complete
• 0.750 g of Kanamycin sulfate were measured and they were diluted in 15 ml of distilled water
• 1.5 g of Ampicillium sodium were weighted and diluted in 15 ml
• The mediums were collocated in the laminar flow cabinet to pour into the petri dishes and striate
• The mediums never solidified
• 2% more of agar was added in the LB broth and the mediums were placed in the autoclave
• The mediums were collocated in the laminar flow cabinet to be poured into the petri dishes and striate
• But the mediums didn’t solidified

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Experiment 5: Preparation of MRS agar mediums (July 19th, 2013)

• The mediums didn’t solidify, because the agar added wasn’t enough
• 10.2 g of MRS broth were measured and 2 g of agar-agar were added
• The pH of the mediums was 6.6
• A stock of HCl at 10% was added until the pH reached 6.4
• The mediums were sterilized with the autoclave for 15 minutes at 121°C
• 40 ml of MRS agar were mixed with 40 ul of kanamycin and poured into 2 petri dishes
• 6 petri dishes were made with only MRS agar
• 2 control petri dishes with MRS agar were striated with L. Plantarum with 1.5 ml of glycerol stock
• The petri dishes were incubated at 37°C for 2 hours

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