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| To test that the lysate is working before doing the Nuclease inactivation, proceed with the Cell-Free Translation of RNA in the Messenger-Dependent Rabbit Reticulocyte Lysate protocol after step 3. There is no need to add mRNA as endogenous message will give a large incorporation of amino acids into protein. |
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1. Thaw 1 ml of lysate (see Hint #1).
2. Add 10 μl of Creatine Kinase Solution and 25 μl 1 mM Haemin.
3. To 800 μl of the supplemented lysate, add 150 μl master mix and mix well.
4. Add 10 μl of 0.1 M CaCl2 and 10 μl of Microccocal Nuclease Solution (about 150 Units)
5. Mix thoroughly and transfer from ice to a ddH2O bath at 20°C for 15 min.
6. Add 20 μl of 0.1 M EGTA and rapidly chill the sample.
7. Divide the messenger-dependent lysate into 50 to 100 μl aliquots and freeze in Liquid Nitrogen.
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| 0.1 M EGTA, neutralized with KOH |
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| Micrococcal Nuclease Solution |
| 1 mg/ml in ddH2O
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| 0.1 M CaCl2 |
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| SOLUTIONS (List Solutions below) |
| Creatine Kinase Solution
Store at -20°C
5 mg/ml Creatine Kinase in 50% aqueous Glycerol
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| Amino Acid Mixture |
| Use stock amino acid solutions of 100 mM. Mix equal volumes of the 19 non-radioactive amino acids. Do not add the amino acid that will be used as the label.
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| Master mix |
| 0.2 M Creatine Phosphate
10 mM MgCl2
333 ul/ml Amino Acid Mixture
2 M KCl
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| 1 mM Haemin |
| 13.26 mg Haemin dissolved in 0.4 ml 0.2 M KOH. Add 0.475 ml ddH2O, 0.125 ml 2 M KCl and 4.0 ml Ethylene Glycol, Centrifuge 5 min at 5000 rpm, Store at -20°C.
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Magnesium Chloride
EGTA
Glycerol
Potassium Chloride
Calcium Chloride
Ethylene Glycol
Micrococcal Nuclease
Creatine Phosphate
Creatine Kinase
Potassium Hydroxide
Haemin
Nitrogen, Liquid
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1. Perform all steps on ice except where noted.
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Pelham, H and Jackson, R. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem 1976; 67:247-56
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