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Preparation of growth mediums (MRS and LB)

Preparation of MRS broth and glycerol stocks

Weight 0.7679 g of powder for MRS broth
Measure 15ml of distilled water in a 100ml measuring cylinder
Weight 10.009g of powder for MRS broth
Dilute it in 100ml of distilled water
Add 100ml of distilled water
Dilute 0.7679g of powder for MRS broth in 15ml of distilled water
Mark the flasks with autoclave tape and sterilize them for 15 minutes at 121⁰c
Label the broth
Mix the 15ml of MRS broth and the 15ml of glycerol to 100% in the laminar flow cabinet. Shake them gently until a homogenous mixture is obtained in a falcon tube of 45ml
Add an aliquot of L. Plantarum to the broth and glycerol solution and seal it with parafilm
Incubate it at 37⁰c and 200rpm
After 24 hours, take the sample out of the incubator to striate it
Place 2500 (x2)ml in 22 eppendorf tubes and using a micropipette of 200µl , cultivate and recompile the mixture of L. Plantarum contained in the falcon tube
Centrifuge 1 eppendorf tube at 13.4 rpm for 3 minutes
Discard the supernatant and add a solution of MRS broth (500ml)
Once the mixture is made, striate two petri dishes with MRS Agar in the laminar flow cabinet
Place the petri dishes facing down in the incubator at 37⁰c and 200 rpm
Store the remaining samples (21 falcon tubes) in deep freezing
Striate E. Coli K12 in two petri dishes with LB Agar
Place the dishes in the incubator at 37⁰c.

Plasmid purification

Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm for 1 minute, two times
Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth
Centrifuge the eppendorf tube for 15min at 6000 rpm
Discard the supernatant
Resuspend the pellet in 250ml resuspension buffer
Add 250ml of Lysis Buffer L7
Gently mix the tube carefully inverting it 5 times carefully
Add and mix softly 350ml of precipitation Buffer N4, inverting the tube
Centrifuge it at 12000 rpm for 10 minutes
Transfer the supernatant into a spin column inside a washtube
Centrifuge it at 12000 rpm for a minute
Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column
Incubate it for one minute at room temperature
Centrifuge the column at 12000 rpm for 1 minute
Discard the liquid from the washtube and place the column inside the tube
Add 700ml of Wash Buffer W9 with ethanol to the column
Centrifuge the column with the washtube at 12000 rpm for 1 minute
Discard the liquid from the washtube
Centrifuge the column with the washtube at 12000 rpm for 1 minute
Discard the liquid from the washtube
Place the column inside an eppendorf tube of 1.5ml
Add 75ml of preheated TE Buffer at the center of the column
(Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes)
Incubate the column for 1 minute at room temperature
The column was centrifuged at 12000 rpm for 2 minutes
(The eppendorf tube contains the purified plasmid)

Preparation of TE buffer

Preparation of competent E. Coli cells

Prepare a falcon tube with 50 ml of CaCl2 0.1M
Prepare another falcon tube with CaCl2 0.1M/ 15% glycerol
Fill 4 eppendorf tubes of 1.5 ml with the E. Coli culture
- 2 of them with 1.5 ml each of E. Coli culture in LB broth (1)
- 2 of them with 1.5 ml each of E. Coli culture in LB broth (2)
Leave the 4 tubes in ice for 10 minutes
Centrifuge the 4 tubes for 3 minutes at 6000 rpm and discard the supernatant
Add 1.5 ml of the culture to each tube
Centrifuge the tubes for 3 minutes at 6000 rpm
Discard the supernatant
gently resuspend the pellet with 1.2 ml CaCl2 0.1M for each tube
Incubate the 4 tubes in ice for 20 minutes
Centrifuge the tubes for 3 minutes at 6000 rpm
Discard the supernatant
Resuspend the pellets with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
Pour them into micro-tubes (300 µl/tube), seal them and freeze them at -80⁰c

Electrophoresis gel for L. Plantarum

Agarose preparation

Prepare TAE 1x with 392 ml of distilled water and 8 ml of TAE 50x (for a 400 ml solution)
Add 0.35 g to 35 ml (grams of agarose)

Preparation of the sample to be placed in the gel

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

Running the gel

Pour the agarose mixture into the casting tray for 35 ml gels with their respective well combs
Remove the well combs once it has solidified
Place the gel in the gel box, with the wells on the side of the anode
Fill the box with TAE 1x up to the indicated measure (equal quantities on both sides of the box)
Pour 15 µl of gene ruler in the first well with a micro pipet
Pour 15 µl of each sample in consecutive wells with a micro pipet
Close the electrophoresis box and connect the power line
Set the current to 100 V and leave it running for 55 minutes
Turn off the current and remove the gel from the chamber
Leave the gel rocking in Ethidium bromide for 15 minutes (always use gloves when handling Ethidium bromide)
Take the gel out of the platform and of the Ethidium bromide and place it in the transiluminator
Observe the results with UV light

Rehydration of plasmids in kit

Number the plate kits as indicated in the iGEM’s parts registry and the plasmids to be used
Pierce the protective layer with a micro-pipet
Rehydrate each plasmid with 10 µl of distilled water
Pipet the liquid until it is homogeneous
It was left resting for 5 minutes to ensure its rehydration