Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/10 - Revision history

Caroline Hom: /* cDNA */

2013-01-11T01:52:23Z

cDNA

←Older revision		Revision as of 01:52, 11 January 2013
Line 9:		Line 9:
	'''Preparation'''		'''Preparation'''
	* Work with samples on ice		* Work with samples on ice
-	* Ice ~~RNase~~: H, SS III RT, and RNase out	+	* Ice: RNase H, SS III RT, and RNase out
	* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT		* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
	'''Sample Reading'''		'''Sample Reading'''

Caroline Hom: /* cDNA */

2013-01-11T01:52:02Z

cDNA

←Older revision		Revision as of 01:52, 11 January 2013
Line 12:		Line 12:
	* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT		* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
	'''Sample Reading'''		'''Sample Reading'''
-	* Add 2u of each sample plus blank to the plate reader	+	* Add 2μL of each sample plus blank to the plate reader
	# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)		# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)
	# K562 Sample 2: 0.385, 2.035, 308.3		# K562 Sample 2: 0.385, 2.035, 308.3

Caroline Hom: /* cDNA */

2013-01-11T01:51:29Z

cDNA

←Older revision		Revision as of 01:51, 11 January 2013
Line 17:		Line 17:
	# SK-N-SH Sample 1: 0.054, 1.819, 43.3		# SK-N-SH Sample 1: 0.054, 1.819, 43.3
	# SK-N-SH Sample 2: 0.201, 2.016, 160.7		# SK-N-SH Sample 2: 0.201, 2.016, 160.7
-	*Need to make ~~8uL~~ solutions of the lowest concentration for each cell line	+	*Need to make 8μL solutions of the lowest concentration for each cell line
-	# Sample 1: 7.~~1uL~~ cells + 0.9 H2O = 2.5ug	+	# Sample 1: 7.1μL cells + 0.9 H2O = 2.5ug
-	# Sample 2: ~~8uL~~ cells = 2.5ng	+	# Sample 2: 8μL cells = 2.5ng
-	# Sample 3: ~~8uL~~ cells = 0.35ug	+	# Sample 3: 8μL cells = 0.35ug
-	# Sample 4: 2.~~2uL~~ cells + H2O = 0.35ng	+	# Sample 4: 2.2μL cells + H2O = 0.35ng
	'''Part 1'''		'''Part 1'''
	* Make sure all samples are filled up to 8uL (fill rest with water if not)		* Make sure all samples are filled up to 8uL (fill rest with water if not)
-	* Add ~~1uL~~ of primer (Oligo dT)	+	* Add 1μL of primer (Oligo dT)
-	* Add 1u of dNTP	+	* Add 1μL of dNTP
	* Incubate for 5 min @ 65°C		* Incubate for 5 min @ 65°C
	* Ice for 1 min		* Ice for 1 min
	'''Part 2'''		'''Part 2'''
-	* Make a MM for 4 rxns: need ~~2uL~~ RT Buffer, ~~4uL~~ MgCl2, ~~2uL~~ DTT, ~~1 uL~~ RNaseOut, and ~~1uL~~ SS III per rxn	+	* Make a MM for 4 rxns: need 2μL RT Buffer, 4μL MgCl2, 2μL DTT, 1μL RNaseOut, and 1μL SS III per rxn
-	* Add ~~10uL~~ of MM to each rxn tube	+	* Add 10μL of MM to each rxn tube
	* Mix tubes gently, centrifuge for a few seconds		* Mix tubes gently, centrifuge for a few seconds
	'''Part 3'''		'''Part 3'''
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	# Stage 2: 5 min @ 85°C		# Stage 2: 5 min @ 85°C
	# Stage 3: Infinity @ 4°C		# Stage 3: Infinity @ 4°C
-	* Add ~~1uL~~: of RNase H	+	* Add 1μL: of RNase H
	* Incubate @ 37°C for 20 min		* Incubate @ 37°C for 20 min
	* Store at -20°C		* Store at -20°C

Caroline Hom: /* Entry title */

2013-01-11T01:48:54Z

Entry title

←Older revision		Revision as of 01:48, 11 January 2013
Line 6:		Line 6:
	\| colspan="2"\|		\| colspan="2"\|
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-	==~~Entry title~~==	+	==cDNA==
-	* ~~Insert content here~~...	+	'''Preparation'''
-		+	* Work with samples on ice
-		+	* Ice RNase: H, SS III RT, and RNase out
		+	* Room temp: Oligo nT, RT Buffer, MgCl2, dNTP mix, water, DTT
		+	'''Sample Reading'''
		+	* Add 2u of each sample plus blank to the plate reader
		+	# K562 Sample 1: 0.44 (260), 2.043 (260/280), 352.1 (ng/mL)
		+	# K562 Sample 2: 0.385, 2.035, 308.3
		+	# SK-N-SH Sample 1: 0.054, 1.819, 43.3
		+	# SK-N-SH Sample 2: 0.201, 2.016, 160.7
		+	*Need to make 8uL solutions of the lowest concentration for each cell line
		+	# Sample 1: 7.1uL cells + 0.9 H2O = 2.5ug
		+	# Sample 2: 8uL cells = 2.5ng
		+	# Sample 3: 8uL cells = 0.35ug
		+	# Sample 4: 2.2uL cells + H2O = 0.35ng
		+	'''Part 1'''
		+	* Make sure all samples are filled up to 8uL (fill rest with water if not)
		+	* Add 1uL of primer (Oligo dT)
		+	* Add 1u of dNTP
		+	* Incubate for 5 min @ 65°C
		+	* Ice for 1 min
		+	'''Part 2'''
		+	* Make a MM for 4 rxns: need 2uL RT Buffer, 4uL MgCl2, 2uL DTT, 1 uL RNaseOut, and 1uL SS III per rxn
		+	* Add 10uL of MM to each rxn tube
		+	* Mix tubes gently, centrifuge for a few seconds
		+	'''Part 3'''
		+	* RT-PCR Machine, set up as follows:
		+	# Stage 1: 50 min @ 50°C
		+	# Stage 2: 5 min @ 85°C
		+	# Stage 3: Infinity @ 4°C
		+	* Add 1uL: of RNase H
		+	* Incubate @ 37°C for 20 min
		+	* Store at -20°C
	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
	\|}		\|}

Caroline Hom: Autocreate 2013/01/10 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2

2013-01-11T01:21:13Z

Autocreate 2013/01/10 Entry for Haynes_Lab:Notebook/Synthetic_Biology_and_Bioinformatics_for_Predictable_Control_of_Therapeutic_Gene2

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Entry title==
* Insert content here...


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