Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/23 - Revision history

Vi Nguyen: /* November 23, 2012 */

2012-12-08T14:18:11Z

November 23, 2012

←Older revision		Revision as of 14:18, 8 December 2012
Line 58:		Line 58:
	Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101		Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101

		+	[[Image:VN_H2B_and_LOV_Transformation_Trial_2.png‎\|thumb\|frame\|center\|There were no cultures seen the next day.]]

	'''Digestion'''		'''Digestion'''

Vi Nguyen: /* November 23, 2012 */

2012-12-08T13:38:37Z

November 23, 2012

←Older revision		Revision as of 13:38, 8 December 2012
Line 60:		Line 60:

	'''Digestion'''		'''Digestion'''
-	* Check ligations from ~~November~~ 20 ~~ligations~~ to see if any colonies contained the insert	+	* Check ligations from 11/20/12 to see if any colonies contained the insert
	* Used 4 colonies from H2B, 4 colonies from LOV plates		* Used 4 colonies from H2B, 4 colonies from LOV plates
	* Each colony liquid culture followed the digestion table accordingly		* Each colony liquid culture followed the digestion table accordingly
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	\|-		\|-
	\| PstI \|\| 0 \|\| 1.0		\| PstI \|\| 0 \|\| 1.0
		+	\|-
	\| 10x buffer \|\| 1.5 \|\| 1.5		\| 10x buffer \|\| 1.5 \|\| 1.5
		+	\|-
	\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5		\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5
		+	\|-
	\| Total vol. \|\| 15.0 \|\| 15.0		\| Total vol. \|\| 15.0 \|\| 15.0
		+	\|-
	\|}		\|}

Vi Nguyen: /* November 23, 2012 */

2012-12-08T13:37:31Z

November 23, 2012

←Older revision		Revision as of 13:37, 8 December 2012
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	\| EcoRI \|\| 1.0\|\| 0		\| EcoRI \|\| 1.0\|\| 0
	\|-		\|-
-	\| PstI \|\| 0 \|\| 1.0 \|-	+	\| PstI \|\| 0 \|\| 1.0
	\| 10x buffer \|\| 1.5 \|\| 1.5		\| 10x buffer \|\| 1.5 \|\| 1.5
	\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5		\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5

Vi Nguyen: /* November 23, 2012 */

2012-12-08T13:36:40Z

November 23, 2012

←Older revision		Revision as of 13:36, 8 December 2012
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	<center>		<center>
-	[[Image:VN_Gel_11-23-12.png~~\|200px~~\|Results of from the digests are shown.]]	+	[[Image:VN_Gel_11-23-12.png\|Results of from the digests are shown.]]
	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]		[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
	</center>		</center>

Vi Nguyen: /* Entry title */

2012-12-08T13:35:33Z

Entry title

←Older revision		Revision as of 13:35, 8 December 2012
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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~~==	+	==November 23, 2012==
-	* Insert ~~content here~~...	+	'''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
		+
		+
		+	'''Ligation'''
		+	* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3'''
		+	* Use 3x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least '''0.8 μL H2B'''
		+	* Use 3x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least '''0.5 μL LOV'''
		+
		+
		+	{\| 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
		+
		+
		+	'''Digestion'''
		+	* Check ligations from November 20 ligations to see if any colonies contained the insert
		+	* Used 4 colonies from H2B, 4 colonies from LOV plates
		+	* Each colony liquid culture followed the digestion table accordingly
		+
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| Reagent \|\| H2B\|\| LOV
		+	\|-
		+	\| DNA \|\| 5.0 \|\| 5.0
		+	\|-
		+	\| EcoRI \|\| 1.0\|\| 0
		+	\|-
		+	\| PstI \|\| 0 \|\| 1.0 \|-
		+	\| 10x buffer \|\| 1.5 \|\| 1.5
		+	\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5
		+	\| Total vol. \|\| 15.0 \|\| 15.0
		+	\|}
		+
		+	* Run entire 15 μL on 1% gel
		+	* Expect 2 bands for H2B and 3 bands for LOV
		+
		+	<center>
		+	[[Image:VN_Gel_11-23-12.png\|200px\|Results of from the digests are shown.]]
		+	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
		+	</center>
		+
		+	<center>
		+	Results of the enzyme digests are shown, with four H2B bands on the left, and three LOV bands on the right. The gel was loaded with 30 μL samples. The bands are at 2000 bp, confirming the vector self ligated and there is no insert.
		+	</center>
		+

Vi Nguyen: Autocreate 2012/11/23 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

2012-12-08T13:21:37Z

Autocreate 2012/11/23 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

New page

{|{{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__

←Older revision		Revision as of 13:37, 8 December 2012
Line 72:		Line 72:
	\| EcoRI \|\| 1.0\|\| 0		\| EcoRI \|\| 1.0\|\| 0
	\|-		\|-
-	\| PstI \|\| 0 \|\| 1.0 \|-	+	\| PstI \|\| 0 \|\| 1.0
	\| 10x buffer \|\| 1.5 \|\| 1.5		\| 10x buffer \|\| 1.5 \|\| 1.5
	\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5		\| dH<sub>2</sub>O \|\| 7.5 \|\| 7.5