Imperial/Wet Lab/Protocols/CE1.3
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<br> Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega. | <br> Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega. | ||
Revision as of 12:18, 26 October 2007
Using the commercial E. Coli cell extract
Aims
Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega.
Equipment
- Eppendorf Tubes
- Gilson p20,p200,p1000
Reagents
- Commercial S30 E.coli extract. Including:
- 175µl Amino Acid Mixture Minus Cysteine, 1mM
- 175µl Amino Acid Mixture Minus Methionine, 1mM
- 175µl Amino Acid Mixture Minus Leucine, 1mM
- 450µl S30 Extract, Circular (3 × 150µl)
- 750µl S30 Premix Without Amino Acids
- Nuclease Free water x1ml
- DNA from midiprep/maxiprep
Protocols
- From the commercially available kit, one reaction mixture comprises of
- 2.5ul of two different types of amino acid mixtures (total volume of 5ul)
- 15ul of S30 Extract, Circular
- 20ul of S30 Premix without amino acids
making up 40ul.
- Add the appropriate amount of DNA to the reaction mixture. (For our experiments we use 2ug and 4ug worth of DNA)
- Top up the mixture to 60ul with nuclease free water.
