IGEM:Imperial/2010/Protocol - Revision history

Benjamin Miller at 16:15, 23 September 2010

2010-09-23T16:15:07Z

←Older revision		Revision as of 16:15, 23 September 2010
Line 1:		Line 1:
		+	{{Imperial2010/Header}}
	==Restriction digests==		==Restriction digests==
	Method:		Method:

Kyasha Sri Ranjan: /* SDS-PAGE – Protocol */

2010-09-16T22:37:12Z

SDS-PAGE – Protocol

←Older revision		Revision as of 22:37, 16 September 2010
Line 123:		Line 123:
	''Short for:'' Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis		''Short for:'' Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

-	# Prepare gel	+	# '''Prepare gel''' : Two glass plates are cleaned with ethanol and are fitted into a holder. The separation layer of the gel is prepared first, following the recipe but before and after addition of the last two substances the solution should be inverted. The mixture, that now starts to polymerize, in now pipetted between the glass plates until it reaches the green bar. Around 700µl of ethanol are than added ontop of the gel, which is left to solidify. The separating gel contains 10% acrylamide '''(toxic!)''' that has been polymerized by TEMED. Stacking gel contains less acryamide for wide pores. After the gel has solidified take out the comb. Once solidified the stacking gel can be prepared using a different recipe but same method. Once the ethanol has been removes the solution is poured onto of the gel and the comb inserted. The gel is now left to solidify.
-	Two ~~glas~~ plates are cleaned with ethanol and are fitted into a holder. The separation layer of the gel is prepared first, following the recipe but before and after addition of the last two substances the solution should be inverted. The mixture, that now starts to polymerize, in now pipetted between the glass plates until it reaches the green bar. Around 700µl of ethanol are than added ontop of the gel, which is left to solidify.	+	# '''Load samples''' : The proteins, which have been denatured by SDS, are loaded into the wells.
-	The separating gel contains 10% acrylamide '''(toxic!)''' that has been polymerized by TEMED. Stacking gel contains less acryamide for wide pores. After the gel has solidified take out the comb.	+	# '''Run gels''' : The gel is run at 100V for around 2-3 hours until dye front has reached the bottom of the gel.
-	Once solidified the stacking gel can be prepared using a different recipe but same method. Once the ethanol has been removes the solution is poured onto of the gel and the comb inserted. The gel is now left to solidify.	+	# '''Analysis of results''' : The gel can be analyzed by staining with Coomassie blue (see protocol) or Western Blot (see protocol)
-	# Load samples	+
-	The proteins, which have been denatured by SDS, are loaded into the wells.	+
-	# Run gels	+
-	The gel is run at 100V for around 2-3 hours until dye front has reached the bottom of the gel.	+
-	# Analysis of results	+
-	The gel can be analyzed by staining with Coomassie blue (see protocol) or Western Blot (see protocol)	+

	==Other useful information==		==Other useful information==

Nicolas Kylilis at 13:05, 16 September 2010

2010-09-16T13:05:22Z

←Older revision		Revision as of 13:05, 16 September 2010
Line 72:		Line 72:
	Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight.		Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight.

-	~~==Catechol Assay==~~
-	* Catechol assay is performed in the plate reader on a 96 well plate
-	* Each well must be filled with 100um of solution
-	* Usually use 90ul of cell culture and 10um of catechol solution
-	* Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM.
-	* Always dilute catechol with H2O.
-	* '''Always''' have a '''blank''' of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution
-	* '''Always''' have a '''negative''' of 90ul growing cells and 10ul of H2O.

	==Transformations==		==Transformations==
Line 156:		Line 148:
	===Midipreps===		===Midipreps===
	The QIAGEN HiSpeed Plasmid Midi Kit is used.		The QIAGEN HiSpeed Plasmid Midi Kit is used.
		+
		+	==Catechol Assay==
		+	* Catechol assay is performed in the plate reader on a 96 well plate
		+	* Each well must be filled with 100um of solution
		+	* Usually use 90ul of cell culture and 10um of catechol solution
		+	* Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM.
		+	* Always dilute catechol with H2O.
		+	* '''Always''' have a '''blank''' of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution
		+	* '''Always''' have a '''negative''' of 90ul growing cells and 10ul of H2O.

Nicolas Kylilis: /* Catechol Assay */

2010-09-16T13:04:24Z

Catechol Assay

←Older revision		Revision as of 13:04, 16 September 2010
Line 78:		Line 78:
	* Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM.		* Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM.
	* Always dilute catechol with H2O.		* Always dilute catechol with H2O.
-	* '''Always''' have a blank of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution	+	* '''Always''' have a '''blank''' of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution
-	* '''Always''' have a negative of 90ul growing cells and 10ul of H2O.	+	* '''Always''' have a '''negative''' of 90ul growing cells and 10ul of H2O.

	==Transformations==		==Transformations==

Nicolas Kylilis: /* Catechol Assay */

2010-09-16T13:03:55Z

Catechol Assay

←Older revision		Revision as of 13:03, 16 September 2010
Line 73:		Line 73:

	==Catechol Assay==		==Catechol Assay==
-	*	+	* Catechol assay is performed in the plate reader on a 96 well plate
		+	* Each well must be filled with 100um of solution
		+	* Usually use 90ul of cell culture and 10um of catechol solution
		+	* Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM.
		+	* Always dilute catechol with H2O.
		+	* '''Always''' have a blank of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution
		+	* '''Always''' have a negative of 90ul growing cells and 10ul of H2O.

	==Transformations==		==Transformations==

Nicolas Kylilis at 12:41, 16 September 2010

2010-09-16T12:41:13Z

←Older revision		Revision as of 12:41, 16 September 2010
Line 9:		Line 9:
	#Gel purification can be used to obtain the desired digestion product from the gel.		#Gel purification can be used to obtain the desired digestion product from the gel.

-	~~'''Catechol assay'''~~
-	*

	'''Reaction mixtures:'''		'''Reaction mixtures:'''
Line 73:		Line 71:
	Insert mass (ng) = 6 x (Insert length (bp)/vector length (bp) x Vector mass (ng)		Insert mass (ng) = 6 x (Insert length (bp)/vector length (bp) x Vector mass (ng)
	Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight.		Once the solution is made up, the tubes are vortexed and then spun down for around 10 seconds in a microcentrifuge. The ligation is done at 14°C in a water bath in the cold cabinet, and is left overnight.
		+
		+	==Catechol Assay==
		+	*

	==Transformations==		==Transformations==

Nicolas Kylilis at 12:40, 16 September 2010

2010-09-16T12:40:25Z

←Older revision		Revision as of 12:40, 16 September 2010
Line 8:		Line 8:
	#Use gel electrophoresis to confirm correct digestion.		#Use gel electrophoresis to confirm correct digestion.
	#Gel purification can be used to obtain the desired digestion product from the gel.		#Gel purification can be used to obtain the desired digestion product from the gel.
		+
		+	'''Catechol assay'''
		+	*

	'''Reaction mixtures:'''		'''Reaction mixtures:'''

A. Florian Sessler: added SDS-PAGE protocol

2010-09-03T08:09:28Z

added SDS-PAGE protocol

←Older revision		Revision as of 08:09, 3 September 2010
Line 117:		Line 117:
	==Overnight Cultures==		==Overnight Cultures==
	Tubes containing 5ml of LB medium are inoculated with cells from one colony and then 5μl of antibiotic (for example chloramphenicol) is added. They are then left at 37°C overnight.		Tubes containing 5ml of LB medium are inoculated with cells from one colony and then 5μl of antibiotic (for example chloramphenicol) is added. They are then left at 37°C overnight.
		+
		+	==SDS-PAGE – Protocol==
		+	''Short for:'' Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
		+
		+	# Prepare gel
		+	Two glas plates are cleaned with ethanol and are fitted into a holder. The separation layer of the gel is prepared first, following the recipe but before and after addition of the last two substances the solution should be inverted. The mixture, that now starts to polymerize, in now pipetted between the glass plates until it reaches the green bar. Around 700µl of ethanol are than added ontop of the gel, which is left to solidify.
		+	The separating gel contains 10% acrylamide '''(toxic!)''' that has been polymerized by TEMED. Stacking gel contains less acryamide for wide pores. After the gel has solidified take out the comb.
		+	Once solidified the stacking gel can be prepared using a different recipe but same method. Once the ethanol has been removes the solution is poured onto of the gel and the comb inserted. The gel is now left to solidify.
		+	# Load samples
		+	The proteins, which have been denatured by SDS, are loaded into the wells.
		+	# Run gels
		+	The gel is run at 100V for around 2-3 hours until dye front has reached the bottom of the gel.
		+	# Analysis of results
		+	The gel can be analyzed by staining with Coomassie blue (see protocol) or Western Blot (see protocol)

	==Other useful information==		==Other useful information==

Harriet D. Gliddon: buffers for restriction enzymes

2010-08-20T11:48:56Z

buffers for restriction enzymes

←Older revision		Revision as of 11:48, 20 August 2010
Line 31:		Line 31:
	\|-		\|-
	\| Total: 10/10 \|\| Total: 30µl \|\|		\| Total: 10/10 \|\| Total: 30µl \|\|
		+	\|}
		+
		+	*The buffer depends on the restriction enzymes used:
		+	'''Prefix Insertion:'''
		+
		+	{\| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
		+
		+	\|-
		+	! !! Enzymes used: !! Required buffer: !!
		+	\|-
		+	\| Prefix (insert)\|\| EcoRI & SpeI \|\| Buffer 2
		+	\|-
		+	\| Suffix (vector)\|\| EcoRI & XbaI \|\| EcoRI buffer
		+
		+	\|}
		+
		+
		+	'''Suffix insertion'''
		+	{\| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
		+
		+	\|-
		+	! !! Enzymes used: !! Required buffer: !!
		+	\|-
		+	\| Prefix (vector)\|\| SpeI & PstI \|\| Buffer 2
		+	\|-
		+	\| Suffix (insert)\|\| XbaI & PstI \|\| Buffer3
		+
	\|}		\|}

Harriet D. Gliddon: reaction mixtures

2010-08-20T11:39:14Z

reaction mixtures

←Older revision		Revision as of 11:39, 20 August 2010
Line 8:		Line 8:
	#Use gel electrophoresis to confirm correct digestion.		#Use gel electrophoresis to confirm correct digestion.
	#Gel purification can be used to obtain the desired digestion product from the gel.		#Gel purification can be used to obtain the desired digestion product from the gel.
		+
		+	'''Reaction mixtures:'''
		+
		+	{\| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
		+
		+	\|-
		+	! Required components !! Example !!
		+	\|-
		+	\| 1/20 Enzyme 1\|\| 1.5µl EcoRI \|\|
		+	\|-
		+	\| 1/20 Enzyme 2
		+	\| 1.5µl PstI \| 1.5µl PstI
		+	\|-
		+	\| 1/10 BSA
		+	\| 3µl BSA
		+	\|-
		+	\| 1/10 Buffer* (x 10) \|\|3µl Buffer 4 (x 10)\|\|
		+	\|-
		+	\| X/10 DNA solution \|\| 20µl DNA (pSB1C3) \|\|
		+	\|-
		+	\| 7-X/10 ddH2O \|\| 1µl ddH2O \|\|
		+	\|-
		+	\| Total: 10/10 \|\| Total: 30µl \|\|
		+	\|}

	==Ligations==		==Ligations==