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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 1.5 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)

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 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 27th, 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 27th, 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 27th, 2013)