Imperial/Wet Lab/Protocols/CE1.3
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__NOTOC__ | __NOTOC__ | ||
| - | + | =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 | *Eppendorf Tubes | ||
*Gilson p20,p200,p1000 | *Gilson p20,p200,p1000 | ||
| - | + | ==Reagents== | |
*Commercial S30 E.coli extract. Including: | *Commercial S30 E.coli extract. Including: | ||
**175µl Amino Acid Mixture Minus Cysteine, 1mM | **175µl Amino Acid Mixture Minus Cysteine, 1mM | ||
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*DNA from midiprep/maxiprep | *DNA from midiprep/maxiprep | ||
| - | + | ==Protocols== | |
| - | #From the commercially available kit, one reaction mixture comprises of | + | #From the commercially available kit, one reaction mixture comprises of: |
| - | *2.5ul of two different types of amino acid mixtures (total volume of 5ul) | + | #*2.5ul of two different types of amino acid mixtures (total volume of 5ul) |
| - | *15ul of S30 Extract, Circular | + | #*15ul of S30 Extract, Circular |
| - | *20ul of S30 Premix without amino acids | + | #*20ul of S30 Premix without amino acids |
| - | + | This makes up a total of 40ul. | |
| - | #Add the appropriate amount of DNA to the reaction mixture. (For our experiments we use 2ug and 4ug worth of DNA) | + | #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. | #Top up the mixture to 60ul with nuclease free water. | ||
Latest revision as of 02:13, 27 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
This makes up a total of 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.
