Team:BIOSINT Mexico/Protocols

From 2013.igem.org

(Difference between revisions)
Line 106: Line 106:
*Resuspend the pellets with 0.6 ml of CaCl2 0.1M/ 15% glycerol each
*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
*Pour them into micro-tubes (300 µl/tube), seal them and freeze them at -80⁰c
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 +
 +
----
 +
'''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

Revision as of 01:42, 28 September 2013

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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


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
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