User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/20 - Revision history

Behzad Damadzadeh: /* 11/20/12 */

2012-12-03T20:01:37Z

11/20/12

←Older revision		Revision as of 20:01, 3 December 2012
Line 59:		Line 59:
	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>		\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
	\|-		\|-
-	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| 57.~~344~~\|\| 1.~~829~~	+	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| 1.829 \|\| 57.344
	\|-		\|-
	\| 2. KAH109, (E/P) \|\| --- \|\| 2.354 \|\| 36.072		\| 2. KAH109, (E/P) \|\| --- \|\| 2.354 \|\| 36.072

Behzad Damadzadeh: /* 11/20/12 */

2012-12-03T20:00:47Z

11/20/12

←Older revision		Revision as of 20:00, 3 December 2012
Line 59:		Line 59:
	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>		\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
	\|-		\|-
-	\| 1. pT7CFE1-CHis (E/P) \|\| 57.344 \|\| 1.829 ~~\|\| ---~~	+	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| 57.344\|\| 1.829
	\|-		\|-
-	\| 2. KAH109, (E/P) \|\| --- \|\| 36.~~072~~ \|\| 2.~~354~~	+	\| 2. KAH109, (E/P) \|\| --- \|\| 2.354 \|\| 36.072
	\|-		\|-
-	\| 3. KAH111, (E/P) \|\| --- \|\| 15.~~003~~ \|\| 2.~~238~~	+	\| 3. KAH111, (E/P) \|\| --- \|\| 2.238 \|\| 15.003
	\|-		\|-
-	\| 4. KAH112, (E/P) \|\| --- \|\| 22.~~857~~ \|\| 1.~~862~~	+	\| 4. KAH112, (E/P) \|\| --- \|\| 1.862 \|\| 22.857
	\|-		\|-
-	\| 5. KAH115, (E/P) \|\| --- \|\| 18.~~312~~ \|\| 2.~~096~~	+	\| 5. KAH115, (E/P) \|\| --- \|\| 2.096 \|\| 18.312
	\|-		\|-
-	\| 6. KAH148, (E/P) \|\| --- \|\| 23.~~735~~ \|\| 2.~~389~~	+	\| 6. KAH148, (E/P) \|\| --- \|\| 2.389 \|\| 23.735
	\|-		\|-
-	\| 7. KAH150, (E/P) \|\| --- \|\| 19.~~822~~ \|\| 1.99	+	\| 7. KAH150, (E/P) \|\| --- \|\| 1.99 \|\| 19.822
	\|-		\|-
-	\| 8. KAH151, (E/P) \|\| --- \|\| 19.~~701~~ \|\| 1.90	+	\| 8. KAH151, (E/P) \|\| --- \|\| 1.90 \|\| 19.701
	\|}		\|}

Behzad Damadzadeh: /* 11/20/12 */

2012-12-03T03:14:21Z

11/20/12

←Older revision		Revision as of 03:14, 3 December 2012
Line 36:		Line 36:
	\|- valign="top"		\|- valign="top"
	\| <u>Reagent</u> \|\| <u>Volume</u> \|\|		\| <u>Reagent</u> \|\| <u>Volume</u> \|\|
-	\| rowspan="9" \| <!--- [[Image:~~image~~.~~tif~~\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose --->	+	\| rowspan="9" \| [[Image:PcTF_in-vitro_Expression.png‎ \|thumb\|350px\| 1. KAH109, 2. KAH111, 3. KAH112, 4. KAH115, 5. KAH148, 6. KAH150, 7. KAH151 8. Human Vector PT7CFE1-CHis(3600 bp) Cut with EcoRI and PstI]]
		+	<!--- [[Image:PcTF_in-vitro_Expression.png‎\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
	\|-		\|-
	\| DNA (plasmid) \|\| 25.0 μL		\| DNA (plasmid) \|\| 25.0 μL

Behzad Damadzadeh: /* 11/20/12 */

2012-11-26T19:00:49Z

11/20/12

←Older revision		Revision as of 19:00, 26 November 2012
Line 82:		Line 82:
	* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)		* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)

-	# BD-116 (~~KAH10~~	+	# BD-116 (KAH109)
-	#	+	# BD-117 (KAH111)
-	#	+	# BD-118 (KAH112)
-	#	+	# BD-119 (KAH115)
-	#	+	# BD-120 (KAH148)
-	#	+	# BD-121 (KAH150)
-	#	+	# BD-122 (KAH-151)
-	#	+	# CTRL

	{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->

Behzad Damadzadeh: /* 11/20/12 */

2012-11-26T18:58:42Z

11/20/12

←Older revision		Revision as of 18:58, 26 November 2012
Line 25:		Line 25:
	'''Digest (Fermentas FD)'''<br>		'''Digest (Fermentas FD)'''<br>
	# pT7CFE1-CHis, E/P		# pT7CFE1-CHis, E/P
		+	# KAH109, E/P
		+	# KAH111, E/P
		+	# KAH112, E/P
		+	# KAH115, E/P
		+	# KAH148, E/P
		+	# KAH150, E/P
		+	# KAH151, E/P

	{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>		\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
	\|-		\|-
-	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| --- \|\| ---	+	\| 1. pT7CFE1-CHis (E/P) \|\| 57.344 \|\| 1.829 \|\| ---
		+	\|-
		+	\| 2. KAH109, (E/P) \|\| --- \|\| 36.072 \|\| 2.354
		+	\|-
		+	\| 3. KAH111, (E/P) \|\| --- \|\| 15.003 \|\| 2.238
		+	\|-
		+	\| 4. KAH112, (E/P) \|\| --- \|\| 22.857 \|\| 1.862
		+	\|-
		+	\| 5. KAH115, (E/P) \|\| --- \|\| 18.312 \|\| 2.096
		+	\|-
		+	\| 6. KAH148, (E/P) \|\| --- \|\| 23.735 \|\| 2.389
		+	\|-
		+	\| 7. KAH150, (E/P) \|\| --- \|\| 19.822 \|\| 1.99
		+	\|-
		+	\| 8. KAH151, (E/P) \|\| --- \|\| 19.701 \|\| 1.90
	\|}		\|}
		+


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	* Note: Use 50 ng of vector this time (instead of 20)		* Note: Use 50 ng of vector this time (instead of 20)
	* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)		* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
		+
		+	# BD-116 (KAH10
		+	#
		+	#
		+	#
		+	#
		+	#
		+	#
		+	#

	{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8		\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8
	\|-		\|-
-	\| Insert DNA \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~	+	\|Inser Name \|\| KAH109 \|\| KAH111 \|\| KAH112 \|\| KAH115 \|\| KAH148 \|\| KAH150 \|\| KAH151 \|\| N/A
		+	\|-
		+	\| Insert DNA \|\| 1.3 \|\| 3.12 \|\| 2.05 \|\| 2.12 \|\| 1.7 \|\| 2.03 \|\| 2.05 \|\| 0
	\|-		\|-
-	\| Vector DNA \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~	+	\| Vector DNA \|\| 0.87 \|\| 0.87 \|\| 0.87 \|\| 0.87 \|\| 0.87 \|\| 0.87 \|\| 0.87 \|\| 0.87
	\|-		\|-
	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0		\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0
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	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0		\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~	+	\| dH<sub>2</sub>O \|\| 1.83 \|\| 0.01 \|\| 1.08 \|\| 1.01 \|\| 1.43 \|\| 1.1 \|\| 1.08 \|\| 3.13
	\|-		\|-
	\|   \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL		\|   \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL

Behzad Damadzadeh: /* 11/16/12 */

2012-11-20T21:22:21Z

11/16/12

←Older revision		Revision as of 21:22, 20 November 2012
Line 7:		Line 7:
	<!-- ##### 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. ##### -->

-	=11/16/12=	+	=11/20/12=
-		+
-	*'''[[User:Karmella Haynes\|---Karmella]] 13:51, 16 November 2012 (EST)''': Here is my suggested procedure for trying the PcTF fusion cloning...	+

	----		----

Behzad Damadzadeh: /* Date */

2012-11-20T21:20:32Z

Date

←Older revision		Revision as of 21:20, 20 November 2012
Line 7:		Line 7:
	<!-- ##### 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. ##### -->

-	=~~Date~~=	+	=11/16/12=

-	'''~~List title~~'''	+	*'''[[User:Karmella Haynes\|---Karmella]] 13:51, 16 November 2012 (EST)''': Here is my suggested procedure for trying the PcTF fusion cloning...
-	# ~~List items~~	+
		+	----
		+	'''Assemblies''': Shown as Part/cuts/purified fragment size
		+	# KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
		+	# KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
		+	# KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
		+	# KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
		+	# KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
		+	# KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
		+	# KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
		+
		+	* Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation<br>
		+	* Note: KAH111-KAH151 are all cut and purified already
		+
		+
		+	'''Digest (Fermentas FD)'''<br>
		+	# pT7CFE1-CHis, E/P
		+
		+	{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
		+	\|- valign="top"
		+	\| <u>Reagent</u> \|\| <u>Volume</u> \|\|
		+	\| rowspan="9" \| <!--- [[Image:image.tif\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
		+	\|-
		+	\| DNA (plasmid) \|\| 25.0 μL
		+	\|-
		+	\| 10x buffer \|\| 3.0
		+	\|-
		+	\| EcoRI \|\| 1.0
		+	\|-
		+	\| PstI \|\| 1.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 0
		+	\|-
		+	\|   \|\| 30 μL --> 37°C/ ~30 min.
		+	\|}
		+
		+
		+
		+	'''Measure concentration(s)'''
		+	{\| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
		+	\|-
		+	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
		+	\|-
		+	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| --- \|\| ---
		+	\|}
		+
		+
		+
		+	'''Ligations''' (trouble shooting suggestions)
		+	* Note: Use 50 ng of vector this time (instead of 20)
		+	* Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
		+
		+	{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
		+	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8
		+	\|-
		+	\| Insert DNA \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
		+	\|-
		+	\| Vector DNA \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
		+	\|-
		+	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0
		+	\|-
		+	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
		+	\|-
		+	\|   \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL
		+	\|}
		+
		+
		+	* Add total ligation reaction to 30 μL DH5α-Turbo cells
		+	* Follow routine "fast" transformation procedure
		+	* Plate onto 100 ug/mL Amp agar plates

Behzad Damadzadeh: Autocreate 2012/11/20 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli

2012-11-20T21:19:37Z

Autocreate 2012/11/20 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli

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{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</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>}}
|-
| colspan="2"|


=Date=

'''List title'''
# List items