Imperial/Wet Lab/Protocols/CE1.4
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| + | __NOTOC__ | ||
| - | === | + | =Using the Home-made S30 E. Coli cell extract= |
| + | ==Aims== | ||
| + | Proper experimental amounts for reaction mixtures of the home-made S30 E.coli cell extract. | ||
| + | |||
| + | ==Equipment== | ||
*Eppendorf Tubes | *Eppendorf Tubes | ||
*Gilson p20,p200,p1000 | *Gilson p20,p200,p1000 | ||
| - | + | ==Reagents== | |
*Pyruvate kinase | *Pyruvate kinase | ||
*rNTPs | *rNTPs | ||
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*DNA from midiprep/maxiprep | *DNA from midiprep/maxiprep | ||
| - | + | ==Protocol== | |
#One reaction mixture comprises: | #One reaction mixture comprises: | ||
| - | *Home made S30 - 16.2ul | + | #*Home made S30 - 16.2ul |
| - | *Reaction Buffer- 30ul | + | #*Reaction Buffer- 30ul |
| - | *rNTP's - 1ul | + | #*rNTP's - 1ul |
| - | *Pyruvate Kinase - 3.1ul | + | #*Pyruvate Kinase - 3.1ul |
| - | *DNA - 4ul | + | #*DNA - 4ul |
| - | *ddH<sub>2</sub> - 5.7ul | + | #*ddH<sub>2</sub> - 5.7ul |
#Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water. | #Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water. | ||
Latest revision as of 02:13, 27 October 2007
Using the Home-made S30 E. Coli cell extract
Aims
Proper experimental amounts for reaction mixtures of the home-made S30 E.coli cell extract.
Equipment
- Eppendorf Tubes
- Gilson p20,p200,p1000
Reagents
- Pyruvate kinase
- rNTPs
- S30 cell extract (home made)
- Reaction buffer (home made)
- Commercial S30 cell extract
- Commercial Pre-incubation mix
- Amino Acids
- Minus Cysteine
- Minus Leucine
- DNA from midiprep/maxiprep
Protocol
- One reaction mixture comprises:
- Home made S30 - 16.2ul
- Reaction Buffer- 30ul
- rNTP's - 1ul
- Pyruvate Kinase - 3.1ul
- DNA - 4ul
- ddH2 - 5.7ul
- Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water.
