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-	==Reagents==	+	'''Aims'''
		+	<br> Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega.
		+
		+	===Equipment===
		+	*Eppendorf Tubes
		+	*Gilson p20,p200,p1000
		+
		+	===Reagents===
	*Pyruvate kinase		*Pyruvate kinase
	*rNTPs		*rNTPs
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	**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
-	#*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
-	#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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Revision as of 14:56, 25 October 2007

Aims
Proper experimental amounts for reaction mixtures of S30 E.coli cell extract from Promega.

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

1. Then add 4ug/2ug worth of DNA, before topping it up to a total volume of 60ul with nuclease free water.