Team:BIOINT Mexico/Lab work

From 2013.igem.org

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9 LB 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.

Revision as of 04:41, 27 September 2013

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Lab work



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


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.