Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08 - Revision history

Vi Nguyen: /* February 8, 2013 */

2013-04-18T12:52:59Z

February 8, 2013

←Older revision		Revision as of 12:52, 18 April 2013
Line 60:		Line 60:

	<center>		<center>
-	[[Image:VN_Gel_2-8-13.png\|Results of the digests are shown~~. It was later found that the transparency used was blocking the UV light~~.]]	+	[[Image:VN_Gel_2-8-13.png\|Results of the digests are shown.]]
	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]		[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
	</center>		</center>

	<center>		<center>
-	Results of the PCR product digests are shown~~, with H2B on the left and LOV on the right~~. ~~The gel~~ was ~~loaded with 5 μL samples~~.	+	Results of the PCR product digests are shown. It was later found that the transparency used was blocking the UV light, so no bands could be seen.
	</center>		</center>

Vi Nguyen: /* February 8, 2013 */

2013-04-18T12:51:29Z

February 8, 2013

←Older revision		Revision as of 12:51, 18 April 2013
Line 60:		Line 60:

	<center>		<center>
-	[[Image:~~Gel_2~~-8-13.png\|Results of the digests are shown.]]	+	[[Image:VN_Gel_2-8-13.png\|Results of the digests are shown. It was later found that the transparency used was blocking the UV light.]]
	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]		[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
	</center>		</center>
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	\| PCR Product \|\| 260\|\| 280 \|\| 260/280 \|\| ng/μL		\| PCR Product \|\| 260\|\| 280 \|\| 260/280 \|\| ng/μL
	\|-		\|-
-	\| H2B \|\| ~~fill~~ \|\| ~~fill~~ \|\| ~~fill~~ \|\| ~~fill~~	+	\| H2B \|\| 0.078\|\| 0.043\|\| 1.81 \|\| 77.995
		+	\|-
		+	\| LOV \|\| 0.08 \|\| 0.044 \|\| 1.817 \|\| 80.443
		+	\|-
		+	\| pSB1A3 \|\| 0.023 \|\| 0.014 \|\| 1.727 \|\| 23.386
	\|-		\|-
-	~~\| LOV \|\| fill \|\| fill \|\| fill \|\| fill~~
	\|}		\|}

Vi Nguyen: /* February 8, 2013 */

2013-02-08T12:40:37Z

February 8, 2013

←Older revision		Revision as of 12:40, 8 February 2013
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	* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end		* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
	* Primer design		* Primer design
-	H2B	+	#H2B
		+
	1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG		1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
		+
	H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA		H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

-	LOV	+	#LOV
		+
	LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT		LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
		+
	LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC		LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC

Vi Nguyen: /* February 8, 2013 */

2013-02-08T12:39:47Z

February 8, 2013

←Older revision		Revision as of 12:39, 8 February 2013
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. ##### -->
	==February 8, 2013==		==February 8, 2013==
-	'''Type IIS Assembly	+	'''Type IIS Assembly - Making PCR Products '''
	* Attempt new assembly method because piecewise assembly was unsuccessful		* Attempt new assembly method because piecewise assembly was unsuccessful
	* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end		* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end

Vi Nguyen: /* Entry title */

2013-02-08T12:39:06Z

Entry title

←Older revision		Revision as of 12:39, 8 February 2013
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	\| 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~~==	+	==February 8, 2013==
-	* ~~Insert content here~~...	+	'''Type IIS Assembly
		+	* Attempt new assembly method because piecewise assembly was unsuccessful
		+	* Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
		+	* Primer design
		+	H2B
		+	1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
		+	H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
		+
		+	LOV
		+	LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
		+	LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
		+
		+	* First, resuspend dry primer oligos (in blue-capped tubes) to 100 μM
		+	* Then make working solutions of 10 μM
		+	* Reactions:
		+
		+	# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
		+	# LOV plasmid + LOV fwd + LOV+his/1A3 rev
		+
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| Reagents \|\| H2B \|\| LOV
		+	\|-
		+	\| Plasmid DNA \|\| 0.2 μL \|\| 0.2
		+	\|-
		+	\| primer 1 (10 μM) \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| primer 2 (10 μM) \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| 2x GoTaq mix \|\| 25.0 \|\| 25.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 22.8 \|\| 22.8
		+	\|-
		+	\| Total \|\| 50.0 \|\| 50.0
		+	\|}
		+
		+	PCR program:
		+	* 95°C, 3 min.
		+	* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 1 min.] x35 cycles
		+	* 72°C, 3 min.
		+	* 4°C ∞
		+
		+	{\| class="wikitable" border=0
		+	\|-
		+	\| <u>Expected:</u><br>1. H2B = 410 <br>2. LOV = 461
		+	\| <br>
		+	\|}
		+
		+	<center>
		+	[[Image:Gel_2-8-13.png\|Results of the digests are shown.]]
		+	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
		+	</center>
		+
		+	<center>
		+	Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
		+	</center>
		+
		+	* The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH<sub>2</sub>O
		+
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| PCR Product \|\| 260\|\| 280 \|\| 260/280 \|\| ng/μL
		+	\|-
		+	\| H2B \|\| fill \|\| fill \|\| fill \|\| fill
		+	\|-
		+	\| LOV \|\| fill \|\| fill \|\| fill \|\| fill
		+	\|}

Vi Nguyen: Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

2013-02-08T12:19:24Z

Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

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