Team:TU Darmstadt/Protocols/SamplePreparationEDX

From 2012.igem.org

Preparation of Escherichia Coli for the energy dispersive X-ray spectroscopy (EDX)


For the EDX measument the cells have to be fixed, embedded in a SPURR-embedding medium (resin) and cut in to small rectangles smaller than 100 nm.


Contents

Fixation

NOTE: The pellet must be about 0.25 g and the preparation takes about 4 days


Primary Fixation-step


  • give about 1 ml pre-fixationmixure to your pellet and incubate at room temperature for at least 1 hour

NOTE: you have to centrifuged your pellet after each step (about 30-45 sec at max. rpm)


Washing-step


  • wash your pellet 4 times with about 750 µL 0.1 M Cacodylate-buffer (CaCo) pH 7.4 for at least 15 min at RT


Secondary fixation-step


  • give about 750 µL 0.5 % Osmium in 0.1 M CaCo Puffer pH 7.4 to your pelett and incubate at RT at least 1 hour


washing-step


  • wash your pellet 2 times with about 750 µL 0.1 M CaCo buffer pH 7.4 for at least 10 min and 2 times with bidest. H2O for 10 min

Dehydration

  • give to your pellett about 750µL 30%,50%,90%,100% acetone and incubate each pellet at RT for at least 10 min
  • give to your pelett about 750µL 100% acetone (dried) and incubate 2 times at RT for at least15 min


Infiltration

  • give to your pelett about 500µL 25 % SPURR (in acetone) and incubate at RT for at least 45 min
  • give to your pelett about 500µL 50 % SPURR (in acetone) and incubate at RT for at least 45 min
  • give to your pelett about 500µL 75 % SPURR (in acetone) and incubate at RT for at least 45 min
  • give to your pelett 2 times about 500µL 100 % SPURR (in acetone) and incubate at RT for at least
  • give to your pelett about 500µL 100 % SPURR (in acetone) and incubate at 4 °C overnight.


Polymerisation

NOTE: your pellet have to be on the bottom of the capsule

  • give to your pelett fresh mixed SPURR and fill a part of your pellet into a capsule and incubate at 74°C over night.


Cutting

  • cut your polymererised capsule with a.... in a thicker than 100 nm rectangles
  • your sample ist ready for EDX


Mixtures

Pre-fixation medium


  1. 0.1 M Cocadynaltebuffer pH 7.4 (with acentic acid)(C2H6AsO2Na x 3 H2O Dimethyle arsine acid sodium salt, 214.13 g/mol) for 50 mL 2.14 g Dimethyle arsine acid sodium salt
  2. 25% Glutaralaldehyde solution
  3. 10 % Formalaldehyde solution
  4. ddH2O

´freshly mixed: annul 0.5 g Paraformaldehyde in 5 mL ddH2O and heat up to 60 °C. Add dropwise NaOH until a clear solution occurs.


Amount for 10 mL pre-fixation medium


  1. 5 mL (endconcentration 0.1 M)
  2. 1 mL (endconcentration 2.5 %)
  3. 2 mL (endconcentration 2 %)
  4. 2 mL ddH2O


E.R.L-embedding media (SPURR)


  1. 3,4 epoxycyclohexyl methyl carboxylate 2.5 g
  2. D.E.R. 736 epoxy resin 1.0 g
  3. Nonenyl-succinic acid 6.5 g
  4. Dimetyl-aminomethanol 0.1 g