Team:BIOINT Mexico/Lab work

From 2013.igem.org

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*Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol
*Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol
*4 eppendorf tubes of 1.5 ml were filled  
*4 eppendorf tubes of 1.5 ml were filled  
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**2 of them with 1.5 ml each of E. Coli culture in LB broth (1)
 
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**2 of them with 1.5 ml each of E. Coli culture in LB broth (2)
 
*The 4 tubes were left in ice for 10 minutes
*The 4 tubes were left in ice for 10 minutes
*The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded
*The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded

Revision as of 06:39, 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 (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



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 µl 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



Experiment 6: Growth of E. Coli (July 21st, 2013)

  • No growth was discernible in the petri dishes with E. Coli, which were left incubating. This attributed to the use of an E. Coli sample, which was in refrigeration since 2011
  • The LB Agar medium was heated in an electric stove for approximately 10 minutes
  • A new sample of lyophilized E. Coli sample was opened and a representative sample was extracted to be rehydrated with LB broth
  • Liquid LB agar was poured into 2 petri dishes and they were left to solidify
  • Both dishes were striated with the rehydrated E. Coli
  • Both dishes were left incubating, after being sealed, during 24 hours at 37⁰c



Experiment 7: Glycerol stocks reactivation (July 22nd, 2013)

  • No discernible growth was observed in the L. Plantarum plates after a 72 hours incubation period. After consulting an external source, it was determined that this was due to glycerol stocks used, which weren’t reactivated
  • Two stocks of glycerol were reactivated with MRS broth
  • 10 ml of MRS broth were added to a 1 ml stock in a falcon tube
  • The second stock was poured into another falcon tube, the result was 1.5 ml of stock with 15 ml of MRS broth
  • Both falcon tubes were sealed and left incubating at 37⁰c



Experiment 8: Overnight growth of E. Coli in LB broth (July 22nd, 2013)

  • 5 ml of LB broth were poured into 2 falcon tubes each
  • An E. Coli colony was added to each tube with broth
  • Both tubes were sealed and left incubating at 37⁰c



Experiment 9: Preparation of mediums for lactobacillus and E. Coli (July 23rd, 2013)

  • 15.3 g of MRS and 6 g of bacto agar were weighted to be diluted in 300 ml
  • 17.5 g of LB agar were weighted to prepare 500 ml
  • The MRS and LB agar were autoclaved
  • 1 ml of E. Coli was inoculated in 100 ml of LB agar in a flask of 250 ml
  • 5 g of LB broth were weighted
  • It was autoclaved for 15 minutes at 121⁰c



Experiment 10: Preparation of TE buffer (July 25th, 2013)

  • 6.05 g of TRIS were weighted and diluted in 60 ml of distilled water
  • 9.3041 g of EDTA were weighted and diluted I 60 ml of distilled water
  • The TRIS’ pH was 10.29, therefore HCl was added until the pH reached 8.3
  • The EDTA’s pH was 3.25, therefore NaOH crystals were added until the pH reached 7.79
  • They were sealed and autoclaved for 15 minutes at 121⁰c



Experiment 11: Preparation of competent E. Coli cells (July 26th, 2013)

  • A falcon tube was prepared with 50 ml of CaCl2 0.1M
  • Another falcon tube was prepared with CaCl2 0.1M/ 15% glycerol
  • 4 eppendorf tubes of 1.5 ml were filled
  • The 4 tubes were left in ice for 10 minutes
  • The 4 tubes were centrifuged for 3 minutes at 6000 rpm and the supernatant was discarded
  • 1.5 ml of the aforementioned culture were added to each tube
  • The tubes were centrifuged for 3 minutes at 6000 rpm
  • The supernatant was discarded
  • The pellet was gently resuspended with 1.2 ml CaCl2 0.1M for each tube
  • The 4 tubes were incubated in ice for 20 minutes
  • The tubes were centrifuged again for 3 minutes at 6000 rpm
  • The supernatant was discarded
  • The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
  • They were poured into micro-tubes (300ml/tube), sealed and frozen at -80⁰c



Experiment 12: L. Plantarum plasmid purification (July 26th, 2013)

  • 1.5 ml of L. Plantarum culture in MRS broth were poured into 2 eppendorf tubes, 1.5 ml each
  • Both tubes were centrifuged for 4 minutes at 12000 rpm
  • The supernatant was discarded and another 1.5 ml of culture were added
  • Both tubes were centrifuged for 4 minutes at 12000 rpm
  • The supernatant was discarded
  • 250 ml of resuspension buffer (R3) were added and the pellets were resuspended until the mixture was homogenous
  • The tubes were incubated at room temperature for 15 minutes
  • 250 ml of Lysis buffer (L7) were added to each tube. They were gently mixed
  • The tubes were incubated at room temperature for 5 minutes
  • 350 ml of precipitation buffer (N4) were added
  • Both tubes were vigorously mixed until a homogenous mixture was obtained
  • The tubes were centrifuged at 12000 rpm for 10 minutes
  • The supernatant was poured into a spin column in a wash tube of 2 ml
  • The columns were centrifuged at 12000 rpm for 1 minute
  • The supernatant was discarded and the column was placed in the new washtube
  • 500 ml of wash buffer (W10) with ethanol were added to the column
  • The columns were incubated for 1 minute at room temperature
  • The columns were centrifuged at 12000 rpm for 1 minute
  • The liquid that went through the columns was discarded
  • 700 ml of wash buffer (W9) were added
  • The columns were centrifuged at 12000 rpm for 1 minute
  • The liquid that went through the columns was discarded
  • The columns were centrifuged at 12000 rpm for 1 minute
  • The liquid that went through the columns and the washtube was discarded
  • The columns were placed in eppendorf tubes of 1.5 ml
  • 70 ml of TE buffer (previously heated at 65⁰c for 3 minutes) were added in the center of each column
  • The columns were incubated at room temperature for 1 minute
  • The columns were centrifuged at 12000 rpm for 2 minutes
  • The eppendorf tubes contain the purified plasmids
  • The columns were removed, the eppendorf tubes were sealed and they were refrigerated at 4⁰c until the next day



Experiment 13: Electrophoresis gel for L. Plantarum (July 27th, 2013)