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Latest revision as of 04:05, 28 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 diluted 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 a 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 in 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 23rd, 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 24th, 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 26th, 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 in 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 28th, 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 28th, 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 29th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)

Preparation of the sample to be placed in the gel

10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml
5 µl of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample

Running the gel

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
15 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 15 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
No sample presented marks in the gel, only the gene ruler

Experiment 14: Overnight culture of L. Plnatarum and agarose preparation (July 29th, 2013)

2 colonies with one day of incubation were inoculated in MRS broth out of a 10 ml sample
They were placed in falcon tubes and left incubating at 37.2⁰c
35 ml of a TAE 1x solution were used to prepare agarose gel for electrophoresis. 0.35 g of agarose were added to the solution to solidify it
The liquid gel was poured into a casting tray and the well combs were placed

Experiment 15: Rehydration of plasmids in kit plates (July 30th, 2013)

The plate kits were numbered as indicated in the iGEM’s parts registry and the plasmids to be used were localized
The protective layer was pierced with a micro-pipet
Each plasmid was rehydrated with 10 µl of distilled water
The liquid was pipetted until it was homogeneous
It was left resting for 5 minutes to ensure its rehydration

Experiment 16: Electrophoresis gel for kit plates plasmids (July 30th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to form a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)
The agarose with TAE 1x was heated in the microwave for 4 seconds

Preparation of the sample to be run in gel

5 µl of each rehydrated plasmid were placed in eppendorf tubes
10 µl of 6x DNA Loading Dye were added and it was pipetted until the mixture was homogeneous

Running the gel

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
15 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 15 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
Thin marks can be appreciated in samples psB4A5 and psB1A7

Experiment 17: E. Coli transformation with linearized plasmids (psB4k5, psB1k3, psB2k3) (July 31st, 2013)

The competent cells were taken out of deep freeze and were left to defrost in ice
They were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube
10 µl of linearized plasmid were added (psB4k5)
It was mixed gently and left incubating in ice for 30 minutes
The tube was put through heat shock in water at 42⁰c for 45 seconds
The tube was placed in ice for 2-5 minutes
900 µl of S.O.C. were added
The tube was left incubating for 1 hour at 37⁰c and 200 rpm
A dish with LB agar and kanamycin was striated with the transformed E. Coli
The dish was sealed and left incubating at 37⁰c
The same procedure was followed for plasmids psB1k3 and psB2k3

Experiment 18: Preparation of transformation buffer CaCl2 50mM (August 2nd, 2013)

1 ml of CaCl2 buffer 200mM were placed in a beaker
3 ml of sterile distilled water were added
With a syringe, it was filled to its maximum capacity with the buffer
A ministart 0.2µm filter was connected to the syringe
The content of the syringe was poured into a sterile falcon tube through the tube
The tube was left cooling in ice

Experiment 19: E. Coli transformation with pGLO (GFP) (August 3rd, 2013)

The competent cells were taken out of deep freeze and left defrosting in ice
They were gently mixed and 50 µl were placed in a cold 1.5 ml eppendorf tube
10 µl of pGLO were added
They were gently mixed and left incubating in ice for 30 minutes
The tube went through heat shock in water at 42⁰c for 45 seconds
The tube was left in ice for 5 minutes
900 µl of S.O.C. was added to the tube
It was gently mixed and left incubating for 1 hour at 37⁰c and 150 rpm
A control dish and a dish with antibiotic were striated
Both dishes were sealed and left incubating facing down at 37⁰c

Experiment 20: MRS and LB mediums preparation (August 5th, 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 for 15 minutes at 121⁰c
They were left in refrigeration

Results:

After an incubating period of 24 hours both, the control and the L-arabinose and ampicillin, plates were observed. Both plates presented bacterial growth. The bacteria in the L-arabinose plate were analyzed in the transilluminator and they glowed. Therefore, the transformation was successful and the gen was expressed correctly. Likewise, it was demonstrated that the previously prepared competent cells are effective.

Experiment 21: Preparation of plates with LB agar and antibiotic (August 6th, 2013)

The LB agar was heated in the electrical stove until it was completely liquefied
The next plates were prepared:
- 1 control (only agar)
- 2 with kanamycin
- 1 with kanamycin and L-arabinose
- 1 with ampicillin
Plate with kanamycin
- 40 µl of kanamycin and 40 ml of LB agar were poured into a beaker (for 2 dishes)
- They were mixed and poured into 2 dishes
Plate with kanamycin and L-arabinose
- 20 µl of kanamycin, 600 µl of L-arabinose and 20 ml of LB agar were mixed in a beaker
- The solution was poured into a petri dish
Plate with ampicillin
- 20 µl of ampicillin and 20 ml of agar were mixed in a beaker
- The solution was poured into a dish
The mediums were prepared again to duplicate the amount of dishes
The plates solidified and they were properly labeled
A total of 9 dishes were prepared
- 1 with LB agar
- 4 with kanamycin
- 2 with kanamycin and L-arabinose
- 2 with ampicillin

Experiment 22: E. Coli transformation with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 7th, 2013)

The DNA of each plasmid was rehydrated with 10 µl of sterile distilled water
Each well of the kit plates was pierced with a micro pipet
Each rehydrated DNA was stored in eppendorf tubes
The competent cells were taken out of deep freeze and were left to defrost in ice
The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube
2 µl of linearized plasmid were added and pipetted until it was homogeneous
It was sealed and left incubating in ice for 30 minutes
The tube was put through heat shock in water at 42⁰c for 60 seconds
The tube was placed in ice for 5 minutes
200 µl of S.O.C. were added
The tube was left incubating for 2 hours at 37⁰c
(This was done with each plasmid)
The samples were striated with a Digralsky spreader in dishes with their correspondent antibiotic and/or inducer
For each plasmid it was inoculated:

1. In a dish, 20µl of a sample of transformed E. Coli (with antibiotics)
2. In a dish, 100 µl of the same transformed E. Coli (with antibiotics)
3. In a control dish, the rest of the transformed cells

they were sealed and left incubating at 37⁰c

Experiment 23: Preparation of competent cells with Lactobacillus Plantarum (August 8th, 2013)

2 eppendorf tubes of 1.5 ml were filled with L. Plantarum in MRS broth
The tubes were left in ice for 10 minutes
The 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 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 24: Purification of L. Plantarum plasmids (August 9th, 2013)

2 different samples of L. Plantarum were used, one from an isolated colony and another form a reactivated stock
1.5 ml of each sample were poured into eppendorf tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
Another 1.5 ml of the samples were added to the tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous
The tubes were incubated for 15 minutes
250 µl of Lysis buffer (L7) were added to each tube and mixed gently
They were left incubating for 5 minutes
350 µl of precipitation buffer were added and it was vigorously shaken
Both tubes were centrifuged at 12000 rpm for 10 minutes
It was observed that before centrifuging the samples, the tube with the reactivated cells had a much larger volume than the other, which meant a mistake was probably made when pipetting
The supernatant was transferred to a spin column in a 2 ml washtube
The tubes were centrifuged for 1 minute at 12000 rpm
The liquid that passed through was discarded and the column was placed again in the washtube
500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute
Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded
700 µl of Wash buffer (W9) were added
Both columns were centrifuged at 12000 rpm for 1 minute
The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml
70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)
The tubes were incubated for 1 minute at room temperature
Both tubes were centrifuged at 12000 rpm for 2 minutes
The columns were discarded and the tubes were sealed and refrigerated at 4⁰c

Experiment 25: Gel for electrophoresis of plasmids (August 9th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)
The agarose with the gel was heated in the microwave for 45 seconds

Preparation of the sample to be placed in the gel

10 µl of each sample of purified plasmid were placed in eppendorf tubes of 1.5 ml
5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample

Running the gel

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
8 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 15 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
Very soft marks were observable for the gene ruler and the L. Plantarum sample

Experiment 26: Preparation of dishes with antibiotics (August 10th, 2013)

The LB agar was heated in the electrical stove until it was completely liquefied
The next plates were prepared:
- 4 control (agar only)
- 4 with kanamycin
- 2 with kanamycin and L-arabinose
- 2 with ampicillin
Plate with kanamycin
- 80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)
- They were mixed and poured into 4 dishes
Plate with kanamycin and L-arabinose
- 40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker
- The solution was poured into 2 petri dishes
Plate with ampicillin
- 40 µl of ampicillin and 40 ml of agar were mixed in a beaker
- The solution was poured into 2 dishes
The mediums were prepared again to duplicate the amount of dishes
The plates solidified, properly labeled and refrigerated

Experiment 27: Transformation of E. Coli with linearized plasmids (psB4k5, psB1A7, psB2k3) (August 10th, 2013)

The competent cells were taken out of deep freeze and were left to defrost in ice
The competent cells were mixed softly and 50 µl of competent cells were placed in a cold eppendorf tube
2 µl of linearized plasmid were added and pipetted until it was homogeneous
It was sealed and left incubating in ice for 30 minutes
The tube was put through heat shock in water at 42⁰c for 60 seconds
The tube was placed in ice for 5 minutes
200 µl of S.O.C. were added
The tube was left incubating for 2 hours at 37⁰c and 150 rpm
(This was done with each plasmid)
Each plasmid was striated in a plate
They were sealed and left incubating at 37⁰c

Results:

After an incubating period of 60 hours the plates with transformed E. Coli, plates were observed. Both plates presented bacterial growth. Therefore, the transformation was successful. Likewise, it was demonstrated that the previously prepared competent cells are effective. However, one dish presented a fungus, which was due to antibiotic which wasn’t filtered. Besides, the plates are not expressing GFP nor RFP

Experiment 28: Preparation of LB broth (August 12th, 2013)

6.0034 g of LB broth were weighted and diluted in 250 ml of distilled water. Afterwards, another 50 ml were added
A cotton and Kraft paper cap was used to cover the flask, and it was autoclaved for 15 minutes at 121⁰c

Experiment 29: Preparation of antibiotics (August 13th, 2013)

1.5 g of ampicillin were diluted in 15 ml of sterile water
0.750 g of kanamycin were weighted and diluted in 15 ml of sterile water
A syringe was used to its maximum capacity to hold the ampicillin stock
A Ministart hydrophilic filter 0.20µm was attached to the syringe
The content of the syringe was filtered into a falcon tube
The same procedure was used for the kanamycin stock
The stocks were sealed and stored in the refrigerator

Experiment 30: Purification of L. Plantarum plasmids (August 15th, 2013)

A sample from an isolated colony was used
1.5 ml of the sample was poured into an eppendorf tube
The tube was centrifuged for 4 minutes at 12000 rpm and the pellet was discarded
Another 1.5 ml of the samples were added to the tube
The tube was centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
250 µl of resuspension buffer (R3) were added and the sample was pipetted until the mixture was homogenous
The tube was incubated for 15 minutes
250 µl of Lysis buffer (L7) was added to the tube and mixed gently
It was left incubating for 5 minutes
350 µl of precipitation buffer were added and it was vigorously shaken
The tube was centrifuged at 12000 rpm for 10 minutes
The supernatant was transferred to a spin column in a 2 ml washtube
The tube was centrifuged for 1 minute at 12000 rpm
The liquid that passed through was discarded and the column was placed again in the washtube
500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute
The tube was centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded
700 µl of Wash buffer (W9) were added
The column was centrifuged at 12000 rpm for 1 minute
The supernatant was discarded and the column was transferred to an eppendorf tube of 1.5 ml
70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)
The tube was incubated for 1 minute at room temperature
The tube was centrifuged at 12000 rpm for 2 minutes
The columns were discarded and the tubes were sealed and refrigerated at 4⁰c

Experiment 31: Gel for electrophoresis of L. Plantarum (purified plasmids) (August 15th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)
The agarose with the gel was heated in the microwave for 45 seconds

Preparation of the sample to be placed in the gel

10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml
5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample

Running the gel

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
15 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 20 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
There was no discernible mark other than the gene ruler

Experiment 32: Purification of L. Plantarum plasmids (August 15th, 2013)

A sample from an isolated colony was used
1.5 ml of the sample was poured into an eppendorf tube, a duplicate was made
The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded
Another 1.5 ml of the samples were added to the tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixture was homogenous
The tubes were incubated for 15 minutes
250 µl of Lysis buffer (L7) were added to the tubes and mixed gently
They were left incubating for 5 minutes
350 µl of precipitation buffer were added and it was vigorously shaken
The tubes were centrifuged at 12000 rpm for 10 minutes
The supernatant was transferred to a spin column in a 2 ml washtube
The tubes were centrifuged for 1 minute at 12000 rpm
The liquid that passed through was discarded and the column was placed again in the washtube
500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute
The tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded
700 µl of Wash buffer (W9) were added
The columns were centrifuged at 12000 rpm for 1 minute
The supernatant was discarded and the columns were transferred to an eppendorf tube of 1.5 ml
70 µl of TE buffer were added to the tube (previously heated at 65⁰c for 3 minutes)
The tube was incubated for 11 minute at room temperature (because the centrifuge was occupied)
The tube was centrifuged at 12000 rpm for 2 minutes
The columns were discarded and the tubes were sealed and refrigerated at 4⁰c

Experiment 33: Purification of transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)

4 samples E. coli from an isolated colony were used
1.5 ml of each sample were poured into eppendorf tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded
Another 1.5 ml of the samples were added to the tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous
The tubes were incubated for 15 minutes
250 µl of Lysis buffer (L7) were added to each tube and mixed gently
They were left incubating for 5 minutes
350 µl of precipitation buffer were added and it was vigorously shaken
The tubes were centrifuged at 12000 rpm for 10 minutes
The supernatant was transferred to a spin column in a 2 ml washtube
The tubes were centrifuged for 1 minute at 12000 rpm
The liquid that passed through was discarded and the column was placed again in the washtube
500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute
Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded
700 µl of Wash buffer (W9) were added
The columns were centrifuged at 12000 rpm for 1 minute
The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml
70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)
The tubes were incubated for 1 minute at room temperature
Both tubes were centrifuged at 12000 rpm for 2 minutes
The columns were discarded and the tubes were sealed and refrigerated at 4⁰c

Experiment 34: Gel for electrophoresis of L. Plantarum plasmids and plasmids (psB3k5, psB4k5, psB1A7, psB2k3) (August 17th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)
The agarose with the gel was heated in the microwave for 45 seconds

Preparation of the sample to be placed in the gel

10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml
5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample

Running the gel

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
15 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 20 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
There are marks in the gel; however most of the samples are blurred. The best defined marks are in track 7, at kbs. These marks are due to a faulty purification

Experiment 35: Purification of L. Plantarum plasmids and transformed E. Coli plasmids (psB3k5, psB4k5, psB1A7, psB2k3, psB1A7) (August 18th, 2013)

A sample of broth with L. Plantarum and LB with E. Coli was used for each corresponding plasmid
1.5 ml of each sample were poured into eppendorf tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the pellet was discarded
Another 1.5 ml of the samples were added to the tubes
The tubes were centrifuged for 4 minutes at 12000 rpm and the supernatant was discarded
250 µl of resuspension buffer (R3) were added and the samples were pipetted until the mixtures were homogenous
The tubes were incubated for 15 minutes
250 µl of Lysis buffer (L7) were added to each tube and mixed gently
They were left incubating for 5 minutes
350 µl of precipitation buffer were added and it was vigorously shaken
The tubes were centrifuged at 12000 rpm for 10 minutes
There was debris floating, so the supernatant was transferred to another tube and it was centrifuged again at 12000 rpm for 10 minutes
The supernatant was transferred to a spin column in a 2 ml washtube
The tubes were centrifuged for 1 minute at 12000 rpm
The liquid that passed through was discarded and the column was placed again in the washtube
500 µl of Wash buffer with ethanol were added and it was left incubating for 1 minute
Both tubes were centrifuged at 12000 rpm for 1 minute and the liquid that went through was discarded
700 µl of Wash buffer (W9) were added
The columns were centrifuged at 12000 rpm for 1 minute
The supernatant was discarded and the columns were transferred to eppendorf tubes of 1.5 ml
70 µl of TE buffer were added to each tube (previously heated at 65⁰c for 3 minutes)
The tubes were incubated for 1 minute at room temperature
Both tubes were centrifuged at 12000 rpm for 2 minutes
The columns were discarded and the tubes were sealed and refrigerated at 4⁰c

Experiment 36: Gel for electrophoresis of purified plasmids (August 18th, 2013)

Agarose preparation

TAE 1x was prepared with 392 ml of distilled water and 8 ml of TAE 50x to prepare a 400 ml solution
A proportion of 80 ml per 0.8 g was made to add 0.35 g to 35 ml (grams of agarose)
The agarose with the gel was heated in the microwave for 45 seconds

Preparation of the sample to be placed in the gel

10 µl of each sample of purified plasmids were placed in eppendorf tubes of 1.5 ml
5 µl of of 6x DNA Loading Dye were added to each one, pipetting until completely homogenizing the sample

Running the gel (x2, because 2 gels were made)

The agarose mixture was poured into the casting tray for 35 ml gels with their respective well combs
It solidified and the well combs were removed
The gel was placed in the gel box, with the wells on the side of the anode
The box was filled with TAE 1x up to the indicated measure
15 µl of gene ruler were placed in the first well
15 µl of each sample were placed in consecutive wells
The electrophoresis box was closed and the power line was connected
The current was set to 100 V and it was left running for 55 minutes
The current was turned off and the gel was removed from the chamber
The gel was left rocking in Ethidium bromide for 20 minutes
The gel was taken out of the platform and of the Ethidium bromide and it was placed in the transiluminator
The results were observed with UV light
Both gels presented intense marks in the tracks correspondent to the purified plasmids of E. Coli in the line of 20 kbs. The gels of L. Plantarum showed no marks

Experiment 37: Preparation of MRS broth, preparation of glycerol stocks and reactivation of glycerol stocks (August 19th, 2013)

10.2 g of MRS broth were weighted
The MRS broth was diluted in 200 ml of distilled water
The mediums were autoclaved for 15 min at 121⁰c
15 ml of MRS broth and 15 ml of glycerol (100%) were mixed in a 45 ml falcon tube, in the laminar flow cabinet
An aliquot of L. Plantarum was added to the solution and it was sealed with parafilm
1 glycerol stock was reactivated with MRS broth
A 1.5 ml stock was used with 15 ml of MRS broth, mixed in a falcon tube
Both tubes were left incubating at 37⁰c and 160 rpm

Experiment 38: Reactivation of the new L. Plantarum strain with glycerol stock (August 19th, 2013)

15 ml of MRS broth were mixed with 15 ml of glycerol (100%) in a 45 ml falcon tube
An aliquot of the new L. Plantarum was added and it was sealed with parafilm

E. Coli transformation with promoter 1,2 and rbs

Take the competent cells out of deep freeze and let them defrost in ice
Gently mix and put 50 µl in a cold 1.5 ml eppendorf tube
Add 10 µl of promoter 1, 2 and rbs
Gently mix them and leave them incubating in ice for 30 minutes
Cause the tube to go through heat shock, placing it in water at 42⁰c for 45 seconds
Cool the tube in ice for 5 minutes
Add 900 µl of S.O.C. to the tube
Gently mix it and leave it incubating for 1 hour at 37⁰c and 150 rpm
Striate a control dish and a dish with antibiotic
Seal both dishes and leave them incubating facing down at 37⁰c

Preparation of dishes with antibiotics (August 10th, 2013) The LB agar was heated in the electrical stove until it was completely liquefied The next plates were prepared: 4 control (agar only) 4 with kanamycin 2 with kanamycin and L-arabinose 2 with ampicillin Plate with kanamycin 80 µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes) They were mixed and poured into 4 dishes Plate with kanamycin and L-arabinose 40 µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker The solution was poured into 2 petri dishes Plate with ampicillin 40 µl of ampicillin and 40 ml of agar were mixed in a beaker The solution was poured into 2 dishes The mediums were prepared again to duplicate the amount of dishes The plates solidified, properly labeled and refrigerated

@@ Line 760: / Line 760: @@
 *Striate a control dish and a dish with antibiotic
 *Seal both dishes and leave them incubating facing down at 37⁰c
+----
+Preparation of dishes with antibiotics (August 10th, 2013)
+The LB agar was heated in the electrical stove until it was completely liquefied
+The next plates were prepared:
+control (agar only)
+with kanamycin
+with kanamycin and L-arabinose
+with ampicillin
+Plate with kanamycin
+µl of kanamycin and 80 ml of LB agar were poured into a beaker (for 2 dishes)
+They were mixed and poured into 4 dishes
+Plate with kanamycin and L-arabinose
+µl of kanamycin, 1200 µl of L-arabinose and 40 ml of LB agar were mixed in a beaker
+The solution was poured into 2 petri dishes
+Plate with ampicillin
+µl of ampicillin and 40 ml of agar were mixed in a beaker
+The solution was poured into 2 dishes
+The mediums were prepared again to duplicate the amount of dishes
+The plates solidified, properly labeled and refrigerated

Team:BIOINT Mexico/Lab work

From 2013.igem.org

Latest revision as of 04:05, 28 September 2013

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