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		+	__NOTOC__
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		+	=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==		==Reagents==
	*Pyruvate kinase		*Pyruvate kinase
Line 9:		Line 21:
	**Minus Cysteine		**Minus Cysteine
	**Minus Leucine		**Minus Leucine
-	*pTet DNA plasmid	+	*DNA from midiprep/maxiprep
-	==Steps==	+	==Protocol==
-	#Fill an eppendorf tube with two samples (56ul x 2) of home made cell extract including reaction buffer, pyruvate kinase and rNTPs.The volumes are shown below:	+	#One reaction mixture comprises:
	#*Home made S30 - 16.2ul		#*Home made S30 - 16.2ul
	#*Reaction Buffer- 30ul		#*Reaction Buffer- 30ul
Line 19:		Line 31:
	#*DNA - 4ul		#*DNA - 4ul
	#*ddH<sub>2</sub> - 5.7ul		#*ddH<sub>2</sub> - 5.7ul
-	#Fill a second eppendorf tube with two samples (40ul x 2) of commercial cell extract including the amino acids mixture, and preincubation buffer.	+	#Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water.
-	#Place a quarter of each mixture in a well in the 96 well plate, making up 2 wells (B3 and B5).	+
-	#In another well (C4), as a control, place another quarter of only the commercial cell extract, with 28ul of water.	+
-	#In the last well (D5), add a quarter of each mixture. This serves as the negative control.	+
-	#In the last well, add nuclease free water (again as a negative control).	+
-	# In wells B3, B5 and C4, add 20ul of DNA.	+
-	#Measure the fluorescence at 30 min intervals for the 1st hour, and hourly intervals thereafter.	+
-	#After each measurement, cover the plate with the sticky lid.	+

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Latest revision as of 02:13, 27 October 2007

• Home
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• Infector Detector
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  • F2620 Comparison
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• Cell by Date
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• Cell-Free
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• Wet Lab
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  • Protocols & Results
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• Dry Lab
  • Modelling
  • Data Analysis
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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

1. One reaction mixture comprises:
   • Home made S30 - 16.2ul
   • Reaction Buffer- 30ul
   • rNTP's - 1ul
   • Pyruvate Kinase - 3.1ul
   • DNA - 4ul
   • ddH₂ - 5.7ul
2. Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water.