Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/06 - Revision history

Vi Nguyen: /* December 6, 2012 */

2013-04-18T12:37:31Z

December 6, 2012

←Older revision		Revision as of 12:37, 18 April 2013
Line 68:		Line 68:
	[[Image:VN_Gibson_Assembly_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]		[[Image:VN_Gibson_Assembly_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]

-	Made more pSB1A3 stock	+	'''Made more pSB1A3 stock'''
	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"		{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
	\|-		\|-

Vi Nguyen: /* December 6, 2012 */

2013-04-18T12:37:01Z

December 6, 2012

←Older revision		Revision as of 12:37, 18 April 2013
Line 68:		Line 68:
	[[Image:VN_Gibson_Assembly_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]		[[Image:VN_Gibson_Assembly_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]

-		+	Made more pSB1A3 stock
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\|Trial \|\| 260 \|\| 280 \|\| 260/280 \|\| ng/μL
		+	\|-
		+	\|1 \|\| 0.062\|\| 0.035 \|\| 1.773\|\| 62.46
		+	\|-
		+	\|2 \|\| 0.052\|\| 0.03 \|\| 1.722\|\| 52.032
		+	\|-
		+	\|3 \|\| 0.082\|\| 0.047\|\| 1.762\|\| 82.222
		+	\|-
		+	\|}

	<!-- ##### 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. ##### -->

Vi Nguyen: /* December 6, 2012 */

2013-04-18T12:32:39Z

December 6, 2012

←Older revision		Revision as of 12:32, 18 April 2013
Line 66:		Line 66:
	Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101		Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101

-	[[Image:~~VN_Gibson_Transformation_Trial_1~~.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]	+	[[Image:VN_Gibson_Assembly_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]

Vi Nguyen: /* December 6, 2012 */

2012-12-08T14:04:50Z

December 6, 2012

←Older revision		Revision as of 14:04, 8 December 2012
Line 66:		Line 66:
	Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101		Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101

		+	[[Image:VN_Gibson_Transformation_Trial_1.png‎\|thumb\|frame\|center\|No colonies were seen the next day.]]

Vi Nguyen: /* December 6, 2012 */

2012-12-08T14:00:21Z

December 6, 2012

←Older revision		Revision as of 14:00, 8 December 2012
Line 58:		Line 58:
	\|-		\|-
	\| Gibson master mix \|\| 15.0 \|\| 15.0 \|\| 15.0		\| Gibson master mix \|\| 15.0 \|\| 15.0 \|\| 15.0
		+	\|-
		+	\| Total volume \|\| 20.0 \|\| 20.0 \|\| 18.0
	\|-		\|-
	\| colspan="3" \| Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.		\| colspan="3" \| Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.

Vi Nguyen: /* December 6, 2012 */

2012-12-08T13:59:27Z

December 6, 2012

←Older revision		Revision as of 13:59, 8 December 2012
Line 38:		Line 38:
	* [final] = 200 ng/μL / 15 μL = ~13 ng/μL		* [final] = 200 ng/μL / 15 μL = ~13 ng/μL

-		+	'''Ligation'''
		+	* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
		+	* Use 1x moles of H2B insert, relative to vector: 1xμL H2B insert = 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 60 ng/μL H2B = at least '''0.07 μL H2B'''
		+	* Use 1x moles of LOV insert, relative to vector: 1xμL LOV insert = 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 62 ng/μL LOV = at least '''0.07 μL LOV'''
		+	* Since the calulated amounts are so small and a volume of 5 μL is needed to add to the Gibson assembly mix, each value was multiplied by 2 and rounded for simplicity

	{\| class="wikitable" width=700px		{\| class="wikitable" width=700px
-	\|   \|\| H2B \|\| LOV \|\| Negative Control \|\| H2B \|\| LOV \|\| ~~Negative Control~~	+	\|   \|\| H2B-LOV \|\| LOV-H2B \|\| Negative Control
		+	\|-
		+	\| H2B-LOV H2B insert DNA (1x mol vector) \|\| 1.0 μL \|\| 0 \|\| 0
		+	\|-
		+	\| H2B-LOV LOV insert DNA (1x mol vector) \|\| 1.0 μL \|\| 0 \|\| 0
	\|-		\|-
-	\| ~~Insert~~ DNA (2x mol vector) \|\| 1.0 μL \|\| 1.0 \|\| ~~--- \|\| 1.~~0 ~~\|\| 1.0 \|\| ---~~	+	\| LOV-H2B H2B insert DNA (1x mol vector) \|\| 0 μL \|\| 1.0 \|\| 0
	\|-		\|-
-	\| ~~Vector~~ DNA (~~50 ng~~) \|\| ~~1.5~~ \|\| 1.5 \|\| ~~1.5 \|\| 1.5 \|\| 1.5 \|\| 1.5~~	+	\| LOV-H2B LOV insert DNA (1x mol vector) \|\| 0 μL \|\| 1.0 \|\| 0
	\|-		\|-
-	\| ~~2x Roche Rapid Ligation buffer~~ \|\| 5.0 \|\| 5.0 \|\| ~~5.0 \|\| 5.0 \|\| 5.0 \|\| 5~~.0	+	\| Vector DNA (39 ng) \|\| 3.0 \|\| 3.0 \|\| 3.0
	\|-		\|-
-	\| ~~T4 ligase~~ \|\| 1.0 ~~NEB~~ \|\| 1.0 ~~NEB~~ \|\| 1.0 ~~NEB \|\| 1.0 Roche \|\| 1.0 Roche \|\| 1.0 Roche~~	+	\| Gibson master mix \|\| 15.0 \|\| 15.0 \|\| 15.0
-	\|-	+
-	~~\| dH<sub>2</sub>O \|\| 1.5 \|\| 1.5 \|\| 2.5 \|\| 1.5 \|\| 1.5 \|\| 2.5~~	+
	\|-		\|-
-	~~\| TOTAL \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0~~	+	\| colspan="3" \| Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 50°C for 1 hour.
-	\|-	+
-	\| colspan="3" \| Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at ~~room temperature~~ for ~~10 minutes~~.	+
	\|}		\|}

Vi Nguyen: /* Entry title */

2012-12-07T00:40:46Z

Entry title

←Older revision		Revision as of 00:40, 7 December 2012
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~~==	+	==December 6, 2012==
-	* Insert ~~content here~~...	+	----
		+
		+	'''Assemblies'''
		+	* Final Gibson assembly products
		+	# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
		+	# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
		+
		+	'''Dephosphorylation''' (11/23/12)
		+
		+	> Dephosphorylation (Roche)
		+	# pSB1A3 - X/S = 2000 bp
		+
		+	{\| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
		+	\|-
		+	\| <u>Reagent</u> \|\| <u>Volume</u>
		+	\|-
		+	\| DNA (clean digest) \|\| 11.0 (~200 ng)
		+	\|-
		+	\| 10x phos. buffer \|\| 1.5
		+	\|-
		+	\| phosphatase \|\| 0.5
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 2.0
		+	\|-
		+	\|   \|\| 15 μL
		+	\|}
		+
		+	* Incubate at 37°C/ 10 min.
		+	* Heat inactivate at 75°C/ 2 min.
		+	* [final] = 200 ng/μL / 15 μL = ~13 ng/μL
		+
		+
		+
		+	{\| class="wikitable" width=700px
		+	\|   \|\| H2B \|\| LOV \|\| Negative Control \|\| H2B \|\| LOV \|\| Negative Control
		+	\|-
		+	\| Insert DNA (2x mol vector) \|\| 1.0 μL \|\| 1.0 \|\| --- \|\| 1.0 \|\| 1.0 \|\| ---
		+	\|-
		+	\| Vector DNA (50 ng) \|\| 1.5 \|\| 1.5 \|\| 1.5 \|\| 1.5 \|\| 1.5 \|\| 1.5
		+	\|-
		+	\| 2x Roche Rapid Ligation buffer \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0 \|\| 5.0
		+	\|-
		+	\| T4 ligase \|\| 1.0 NEB \|\| 1.0 NEB \|\| 1.0 NEB \|\| 1.0 Roche \|\| 1.0 Roche \|\| 1.0 Roche
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 1.5 \|\| 1.5 \|\| 2.5 \|\| 1.5 \|\| 1.5 \|\| 2.5
		+	\|-
		+	\| TOTAL \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0
		+	\|-
		+	\| colspan="3" \| Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes.
		+	\|}
		+
		+	Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
		+
		+

Vi Nguyen: Autocreate 2012/12/06 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

2012-12-07T00:04:02Z

Autocreate 2012/12/06 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

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{|{{table}} width="800"
|-
|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>}}
|-
| colspan="2"|

==Entry title==
* Insert content here...


|}

__NOTOC__