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RECIPES > RRL AND WG RECIPES

> Retic Lysate Reagents

   

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> Wheat Germ Buffers and Reagents

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E mix
0.1M ATP 10 uL
0.1M GTP 10 uL
0.4M CP 25 uL
19 aa -met 40 uL
35S Met (250 uCi) 100 uL
2M Tris base 3 uL or as needed to pH 7
H2O 12 uL
Total 200
CB 20x
  10% linked unlinked
4M KAc 5 mL 5 mL
2M Tris Ac pH 7.5 1 mL 1 mL
1M MgCl2 0.2 mL 0.5 mL
1M DTT - 0.2 mL
H2O 3.8 mL 3.3 mL
Total 10.0 mL 10.0 mL

tRNA 10 mg/mL PIN 200X

10mg/mL chymostatin (DMSO) 20 uL
10mg/mL antipain (DMSO) 20 uL
10mg/mL leupeptin (H2O) 20 uL
10mg/ml pepstatin (H2O) 20 uL
2mg/mL aprotinin (H2O) 200 uL
Water 9.72 mL
Total 10.0 mL

Translation Reaction 10 uL, 10% linked

CB 20X 0.5 uL
E mix 2.0
RRL 4.2
tRNA 10mg/mL 0.1
PIN 200X 0.1
CK 0.1
RNasin 0.1
Water 1.4
8.5 uL
SP6 transcripts 1.0
membranes 0.5
TOTAL 10.0 uL

Incubate 24oC 1 h. Transfer onto ice to stop reaction. 1 uL of this reaction run on SDS-PAGE and fluorographed is usually sufficient to visualize with an overnight exposure.