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CELL FREE SYSTEMS > Wheat Germ Cell-Free Translation

> Protocol for wheat germ cell-free translation

   

> Wheat Germ Extract Buffers and reagents

   

> Sample WG Protocol

   

> Wheat Germ Extract Recipes

   

> Troubleshooting Wheat Germ Translation Reactions

    > Preparation of a wheat germ extract

 

Wheat Germ Extract Recipes

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CB 10X

For 20% WG:
  CB 10X unlinked CB 10X 10% linked Final in reaction
1M Hepes-KOH pH 7.5 120 uL 40 uL 20 mM
4M KOAc 300 300 140 mM
1M MgOAc2 10 - 2.4 mM
0.1M spermidine 40 20 0.4mM
1M DTT 12 2 2 mM
0.5 M EDTA 2 2 0.1 mM
Water 516 636  
Total 1 mL 1 mL  

E mix, tRNA, pin

- same as for RRL
Translation reaction 10 æL, 20% WG, 10% linked
CB 10X 1 uL
E mix 2
WG 2
tRNA 10mg/mL 0.1
PIN 0.1
RNasin 0.1
CK 0.1
Water 3.1
  8.5 uL
Transcripts 1
Membranes 0.5
Total 10 uL

Incubate 24oC 1 h. Transfer onto ice to stop reaction. 1 uL of this reaction run on SDS-PAGE and fluorographed can usually be visualized after an overnight exposure.