Team:BIOSINT Mexico/Protocols

From 2013.igem.org

(Difference between revisions)
Line 75: Line 75:
*The column was centrifuged at 12000 rpm for 2 minutes
*The column was centrifuged at 12000 rpm for 2 minutes
*(The eppendorf tube contains the purified plasmid)
*(The eppendorf tube contains the purified plasmid)
 +
 +
 +
----
 +
'''Preparation of TE buffer'''
 +
 +
*Dilute 6.05 g of TRIS in 60 ml of distilled water
 +
*Dilute 9.3041 g of EDTA in 60 ml of distilled water
 +
*Add HCl until the TRIS’ pH reaches 8.3
 +
*Add NaOH crystals until the EDTA’S pH reaches 7.79
 +
*Seal and autoclave for 15 minutes at 121⁰c
 +
 +
 +
----
 +
'''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

Revision as of 01:25, 28 September 2013

SmartPro

Smartpro

Igem LogotipoDefinitivo



Protocols



Preparation of growth mediums (MRS and LB)

  • Weight 18.6 g of Agar MRS to prepare 300ml of medium
  • Weight 7 g of Agar LB to prepare 200ml of medium
  • Dilute 5 g of A-MRS in 80 ml of distilled water
  • Add 5 g and 170 ml of distilled water
  • Dilute 7 g of Agar LB in 100ml of distilled water
  • Add 100ml of distilled water
  • Place the autoclave tape
  • Set the autoclave
  • When the temperature of the autoclave reaches 120⁰c, cut the power by half
  • When the temperature reaches 105⁰c, open the valve.
  • Retrieve the mediums



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

  • Dilute 6.05 g of TRIS in 60 ml of distilled water
  • Dilute 9.3041 g of EDTA in 60 ml of distilled water
  • Add HCl until the TRIS’ pH reaches 8.3
  • Add NaOH crystals until the EDTA’S pH reaches 7.79
  • Seal and autoclave for 15 minutes at 121⁰c



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