Team:BIOINT Mexico/Lab work
From 2013.igem.org
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'''Experiment 3: Plasmid purification (July 16th, 2013)''' | '''Experiment 3: Plasmid purification (July 16th, 2013)''' | ||
- | *An eppendorf tube of 1. | + | *An eppendorf tube of 1.5 ml 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 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 eppendorf tube was centrifuged for 15min at 6000 rpm | ||
*The supernatant was discarded | *The supernatant was discarded | ||
- | *The pellet was resuspended in | + | *The pellet was resuspended in 250 µl resuspension buffer |
- | * | + | *250 µl of Lysis Buffer L7 |
*The tube was gently mixed inverting it 5 times carefully | *The tube was gently mixed inverting it 5 times carefully | ||
- | * | + | *350 µl of precipitation Buffer N4 were added and mixed softly inverting the tube |
*It was centrifuged at 12000 rpm for 10 minutes | *It was centrifuged at 12000 rpm for 10 minutes | ||
*The supernatant was transferred to a spin column inside a washtube | *The supernatant was transferred to a spin column inside a washtube | ||
*It was centrifuged at 12000 rpm for a minute | *It was centrifuged at 12000 rpm for a minute | ||
- | *The supernatant was discarded and 500 | + | *The supernatant was discarded and 500 µl of Wash Buffer with ethanol (w10) were added to the column |
*It was incubated for one minute at room temperature | *It was incubated for one minute at room temperature | ||
*The column was centrifuged at 12000 rpm for 1 minute | *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 | *The liquid from the washtube was discarded and the column was placed inside the tube | ||
- | * | + | *700 µl 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 column with the washtube was centrifuged at 12000 rpm for 1 minute | ||
*The liquid from the washtube was discarded | *The liquid from the washtube was discarded | ||
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*The liquid from the washtube was discarded | *The liquid from the washtube was discarded | ||
*The column was placed inside an eppendorf tube of 1.5ml | *The column was placed inside an eppendorf tube of 1.5ml | ||
- | * | + | *75 µl 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 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 incubated for 1 minute at room temperature | ||
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*A stock of HCl at 10% was added until the pH reached 6.4 | *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 | *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 | + | *40 ml of agar were mixed with 40 µl of stock of ampicillin and they were poured into 2 petri dishes |
+ | *40 ml of MRS agar were mixed with 40 µl of kanamycin stock and they were poured into 2 petri dishes | ||
*6 petri dishes were made with only MRS agar | *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 | *2 control petri dishes with MRS agar were striated with L. Plantarum with 1.5 ml of glycerol stock | ||
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*The supernatant was discarded | *The supernatant was discarded | ||
*The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each | *The pellets were resuspended with 0.6 ml of CaCl2 0.1M/ 15% glycerol each | ||
- | *They were poured into micro-tubes ( | + | *They were poured into micro-tubes (300 µl/tube), sealed and frozen at -80⁰c |
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*Both tubes were centrifuged for 4 minutes at 12000 rpm | *Both tubes were centrifuged for 4 minutes at 12000 rpm | ||
*The supernatant was discarded | *The supernatant was discarded | ||
- | *250 | + | *250 µl 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 | *The tubes were incubated at room temperature for 15 minutes | ||
- | *250 | + | *250 µl of Lysis buffer (L7) were added to each tube. They were gently mixed |
*The tubes were incubated at room temperature for 5 minutes | *The tubes were incubated at room temperature for 5 minutes | ||
- | *350 | + | *350 µl of precipitation buffer (N4) were added |
*Both tubes were vigorously mixed until a homogenous mixture was obtained | *Both tubes were vigorously mixed until a homogenous mixture was obtained | ||
*The tubes were centrifuged at 12000 rpm for 10 minutes | *The tubes were centrifuged at 12000 rpm for 10 minutes | ||
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*The columns were centrifuged at 12000 rpm for 1 minute | *The columns were centrifuged at 12000 rpm for 1 minute | ||
*The supernatant was discarded and the column was placed in the new washtube | *The supernatant was discarded and the column was placed in the new washtube | ||
- | *500 | + | *500 µl of wash buffer (W10) with ethanol were added to the column |
*The columns were incubated for 1 minute at room temperature | *The columns were incubated for 1 minute at room temperature | ||
*The columns were centrifuged at 12000 rpm for 1 minute | *The columns were centrifuged at 12000 rpm for 1 minute | ||
*The liquid that went through the columns was discarded | *The liquid that went through the columns was discarded | ||
- | *700 | + | *700 µl of wash buffer (W9) were added |
*The columns were centrifuged at 12000 rpm for 1 minute | *The columns were centrifuged at 12000 rpm for 1 minute | ||
*The liquid that went through the columns was discarded | *The liquid that went through the columns was discarded | ||
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*The liquid that went through the columns and the washtube was discarded | *The liquid that went through the columns and the washtube was discarded | ||
*The columns were placed in eppendorf tubes of 1.5 ml | *The columns were placed in eppendorf tubes of 1.5 ml | ||
- | *70 | + | *70 µl 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 incubated at room temperature for 1 minute | ||
*The columns were centrifuged at 12000 rpm for 2 minutes | *The columns were centrifuged at 12000 rpm for 2 minutes |
Revision as of 07:27, 27 September 2013
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 1.5 ml 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 250 µl resuspension buffer
- 250 µl of Lysis Buffer L7
- The tube was gently mixed inverting it 5 times carefully
- 350 µl 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 µl 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
- 700 µl 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
- 75 µl 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 agar were mixed with 40 µl of stock of ampicillin and they were poured into 2 petri dishes
- 40 ml of MRS agar were mixed with 40 µl of kanamycin stock and they were 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 (300 µl/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 µl 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 µl of Lysis buffer (L7) were added to each tube. They were gently mixed
- The tubes were incubated at room temperature for 5 minutes
- 350 µl 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 µl 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 µl 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 µl 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)