User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/16 - Revision history

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

Behzad Damadzadeh — Mon, 26 Nov 2012 20:46:54 GMT

11/16/12

←Older revision		Revision as of 20:46, 26 November 2012
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	'''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 -->
	\|- 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\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose
	\|-		\|-
	\| DNA (plasmid) \|\| 25.0 μL		\| DNA (plasmid) \|\| 25.0 μL
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	\|}		\|}

-	~~# BD-116, (KAH109)~~
-	~~# BD-117, (KAH111)~~
-	~~# BD-118, (KAH112)~~
-	~~# BD-119, (KAH115)~~
-	~~# BD-120, (KAH148)~~
-	~~# BD-121, (KAH150)~~
-	~~# BD-122, (KAH151)~~
-	~~# CTRL~~

	'''Ligations''' (trouble shooting suggestions)		'''Ligations''' (trouble shooting suggestions)
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	\|}		\|}

		+	'''Plates Nomenclature
		+	# BD-116, (KAH109)
		+	# BD-117, (KAH111)
		+	# BD-118, (KAH112)
		+	# BD-119, (KAH115)
		+	# BD-120, (KAH148)
		+	# BD-121, (KAH150)
		+	# BD-122, (KAH151)
		+	# CTRL

	* Add total ligation reaction to 30 μL DH5α-Turbo cells		* Add total ligation reaction to 30 μL DH5α-Turbo cells
	* Follow routine "fast" transformation procedure		* Follow routine "fast" transformation procedure
	* Plate onto 100 ug/mL Amp agar plates		* Plate onto 100 ug/mL Amp agar plates

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

Behzad Damadzadeh — Mon, 26 Nov 2012 20:32:29 GMT

11/16/12

←Older revision		Revision as of 20:32, 26 November 2012
Line 70:		Line 70:
	\|}		\|}

-		+	# BD-116, (KAH109)
		+	# BD-117, (KAH111)
		+	# BD-118, (KAH112)
		+	# BD-119, (KAH115)
		+	# BD-120, (KAH148)
		+	# BD-121, (KAH150)
		+	# BD-122, (KAH151)
		+	# CTRL

	'''Ligations''' (trouble shooting suggestions)		'''Ligations''' (trouble shooting suggestions)

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

Behzad Damadzadeh — Mon, 26 Nov 2012 20:30:36 GMT

11/16/12

←Older revision		Revision as of 20:30, 26 November 2012
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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 */

Behzad Damadzadeh — Mon, 26 Nov 2012 20:29:07 GMT

11/16/12

←Older revision		Revision as of 20:29, 26 November 2012
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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) \|\| --- \|\| 1.829 \|\| 57.344
		+	\|-
		+	\| 2. KAH109 (E/P) \|\| --- \|\| 2.354 \|\| 36.072
		+	\|-
		+	\| 3. KAH111 (E/P) \|\| --- \|\| 2.238 \|\| 15.003
		+	\|-
		+	\| 4. KAH112 (E/P) \|\| --- \|\| 1.862 \|\| 22.857
		+	\|-
		+	\| 5. KAH115 (E/P) \|\| --- \|\| 2.096 \|\| 18.312
		+	\|-
		+	\| 6. KAH148 (E/P) \|\| --- \|\| 2.389 \|\| 23.735
		+	\|-
		+	\| 7. KAH150 (E/P) \|\| --- \|\| 1.99 \|\| 19.822
		+	\|-
		+	\| 8. KAH151 (E/P) \|\| --- \|\| 1.9 \|\| 19.701
	\|}		\|}

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	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8		\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8
	\|-		\|-
-	\| Insert ~~DNA~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~ \|\| ---	+	\| Insert 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

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:22:57 GMT

11/16/12

←Older revision		Revision as of 19:22, 16 November 2012
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	# KAH151/pT7CFE1-CHis: KAH191/(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: KAH109 is being re-done as an internal control to trouble-shoot the ligation<br>	+	* Note: KAH111-KAH151 are all cut and purified already
-	Note: KAH111-KAH151 are all cut and purified already	+

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:21:57 GMT

11/16/12

←Older revision		Revision as of 19:21, 16 November 2012
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	{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
-	\|-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:image.tif\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose --->
	\|-		\|-
	\| DNA (plasmid) \|\| 25.0 μL		\| DNA (plasmid) \|\| 25.0 μL

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:20:38 GMT

11/16/12

←Older revision		Revision as of 19:20, 16 November 2012
Line 32:		Line 32:
	\|-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:image.tif\|350px\|Cloning digest ]]<br>30 μL/lane, 1% agarose
	\|-		\|-
	\| DNA (plasmid) \|\| 25.0 μL		\| DNA (plasmid) \|\| 25.0 μL

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:07:53 GMT

11/16/12

←Older revision		Revision as of 19:07, 16 November 2012
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-	> Digest (Fermentas FD)<br>	+	'''Digest (Fermentas FD)'''<br>
	# pT7CFE1-CHis, E/P		# pT7CFE1-CHis, E/P

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-	> Measure concentration(s)	+	'''Measure concentration(s)'''
	{\| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
	\|-		\|-
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-	> Ligations (trouble shooting suggestions)	+	'''Ligations''' (trouble shooting suggestions)
	* 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)

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:07:16 GMT

11/16/12

←Older revision		Revision as of 19:07, 16 November 2012
Line 78:		Line 78:
	\|   \|\| 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
	\|}		\|}
		+
		+
		+	* Add total ligation reaction to 30 μL DH5α-Turbo cells
		+	* Follow routine "fast" transformation procedure
		+	* Plate onto 100 ug/mL Amp agar plates

Karmella Haynes: /* 11/16/12 */

Karmella Haynes — Fri, 16 Nov 2012 19:03:35 GMT

11/16/12

←Older revision		Revision as of 19:03, 16 November 2012
Line 56:		Line 56:
	\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| --- \|\| ---		\| 1. pT7CFE1-CHis (E/P) \|\| --- \|\| --- \|\| ---
	\|}		\|}
		+


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	{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
-	\|   \|\| 1 \|\| 2 \|\|	+	\|   \|\| 1 \|\| 2 \|\| 3 \|\| 4 \|\| 5 \|\| 6 \|\| 7 \|\| 8
	\|-		\|-
-	\| Insert DNA \|\| ~~3.5~~ \|\| --- \|\|	+	\| Insert DNA \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
	\|-		\|-
-	\| Vector DNA \|\| ~~0.5~~ \|\| ~~0.5~~ \|\|	+	\| Vector DNA \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
	\|-		\|-
-	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\|	+	\| 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 \|\|	+	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| --- \|\| ~~3.5~~ \|\|	+	\| dH<sub>2</sub>O \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| --- \|\| ---
	\|-		\|-
-	\|   \|\| 10 μL \|\| 10 μL \|\|	+	\|   \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL \|\| 10 μL
	\|}		\|}