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Team:BIOINT Mexico/Lab work
From 2013.igem.org
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9 LB Agar | 9 LB Agar | ||
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*E. Coli K12 was striated in two petri dishes with LB Agar | *E. Coli K12 was striated in two petri dishes with LB Agar | ||
*They were placed in the incubator at 37⁰c. | *They were placed in the incubator at 37⁰c. | ||
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*The column was centrifuged at 12000 rpm for 2 minutes | *The column was centrifuged at 12000 rpm for 2 minutes | ||
*The eppendorf tube contains the purified plasmid | *The eppendorf tube contains the purified plasmid | ||
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| + | '''Experiment 4: Preparation of mediums for lactobacillus and E. Coli (April 12, 2013)''' | ||
Revision as of 04:49, 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 (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.
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
Experiment 4: Preparation of mediums for lactobacillus and E. Coli (April 12, 2013)
