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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