Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/05 - Revision history

Vi Nguyen: /* December 5, 2012 */

Vi Nguyen — Sat, 08 Dec 2012 13:46:33 GMT

December 5, 2012

←Older revision		Revision as of 13:46, 8 December 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. ##### -->
	==December 5, 2012==		==December 5, 2012==
-	'''Results from last ~~week's~~ ligation'''	+	'''Results from last 11/23/12 ligation'''
	* None of the colonies on any plates grew		* None of the colonies on any plates grew
	* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method		* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
Line 53:		Line 53:
	* Be sure to elute using 15 μL dH<sub>2</sub>O		* Be sure to elute using 15 μL dH<sub>2</sub>O

		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\|Construct \|\| Product \|\| 260 \|\| 280 \|\| 260/280 \|\| ng/μL
		+	\|-
		+	\|H2B-LOV \|\| H2B 1 \|\| 0.061 \|\| 0.033 \|\| 1.863 \|\| 60.545
		+	\|-
		+	\|H2B-LOV \|\| H2B 2 \|\| 0.056 \|\| 0.03 \|\| 1.857 \|\| 56.22
		+	\|-
		+	\|H2B-LOV \|\| LOV 1 \|\| 0.063 \|\| 0.033 \|\| 1.903 \|\| 62.673
		+	\|-
		+	\|H2B-LOV \|\| LOV 2 \|\| 0.06 \|\| 0.04 \|\| 1.85 \|\| 59.844
		+	\|-
		+	\|LOV-H2B \|\| H2B 1 \|\| 0.072 \|\| 0.033 \|\| 1.807 \|\| 72.381
		+	\|-
		+	\|LOV-H2B \|\| H2B 2 \|\| 0.06 \|\| 0.036 \|\| 1.799 \|\| 59.925
		+	\|-
		+	\|LOV-H2B \|\| LOV 1 \|\| 0.006 \|\| 0.036 \|\| 1.849 \|\| 65.881
		+	\|-
		+	\|LOV-H2B \|\| LOV 2 \|\| 0.065 \|\| 0.035 \|\| 1.839 \|\| 65.285
		+	\|}

	<!-- ##### 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: /* 11/21/12 */

Vi Nguyen — Fri, 07 Dec 2012 00:11:11 GMT

11/21/12

←Older revision		Revision as of 00:11, 7 December 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. ##### -->
	==December 5, 2012==		==December 5, 2012==
-	~~==11/21/12==~~
-
	'''Results from last week's ligation'''		'''Results from last week's ligation'''
	* None of the colonies on any plates grew		* None of the colonies on any plates grew
Line 54:		Line 52:
	* Clean up PCR products using the Zymo Clean and Concentrator kit		* Clean up PCR products using the Zymo Clean and Concentrator kit
	* Be sure to elute using 15 μL dH<sub>2</sub>O		* Be sure to elute using 15 μL dH<sub>2</sub>O
-
-	~~----~~
-
-	*'''[[User:Karmella Haynes\|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
-
-	~~'''Assemblies'''~~
-	~~# NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp~~
-	~~# NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp~~
-	~~# NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp~~
-	~~# Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp~~
-	~~# Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp~~
-	~~# Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp~~
-
-	* Digests:
-	** In a 30 μL reaction, cut H2B with XbaI and SpeI
-	** In a 30 μL reaction, cut LOV with XbaI and SpeI
-
-	~~{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"~~
-	\|-
-	~~\| Reagent \|\| H2B PCR \|\| LOV PCR~~
-	\|-
-	~~\| DNA \|\| 15.0 \|\| 15.0~~
-	\|-
-	~~\| XbaI \|\| 1.0\|\| 1.0~~
-	\|-
-	~~\| SpeI \|\| 1.0 \|\| 1.0~~
-	\|-
-	~~\| 10x buffer \|\| 3.0 \|\| 3.0~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| 10.0 \|\| 10.0~~
-	\|-
-	~~\| Total vol. \|\| 30. 0 \|\| 30.0~~
-	\|}
-
-
-	* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
-	* Cut band out of gel (use UV box)
-
-
-	~~Results from gel recovery:~~
-	~~{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"~~
-	\|-
-	~~\| Sample\|\| 260\|\| 260/280 \|\| ng/μL~~
-	\|-
-	~~\| H2B - X/S 1 \|\| 0.016 \|\|1.807\|\| 15.961~~
-	\|-
-	~~\| H2B - X/S 2 \|\| 0.009 \|\| 1.976 \|\| 8.619~~
-	\|-
-	~~\| LOV - X/S 1 \|\| 0.006\|\| 1.694 \|\| 6.455~~
-	\|-
-	~~\| LOV - X/S 2 \|\| 0.025\|\| 1.883 \|\| 25.363~~
-	\|}
-
-
-	~~[[Image:VN_Gel_11-21-12.png\|Results of from the digests are shown.]]~~
-	~~[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]~~
-
-	~~# H2B = 410~~
-	~~# LOV = 461~~
-
-	* There were two samples made of each PCR reaction. The two from the H2B are shown on the left, and the two from the LOV are shown on the right.
-
-
-	~~'''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~~
-

Vi Nguyen: /* 11/21/12 */

Vi Nguyen — Wed, 05 Dec 2012 23:51:53 GMT

11/21/12

←Older revision		Revision as of 23:51, 5 December 2012
Line 17:		Line 17:
	* Note: Primers add a XbaI site upstream and the SpeI site downstream		* Note: Primers add a XbaI site upstream and the SpeI site downstream
	* Final Gibson assembly products		* Final Gibson assembly products
-	# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end	+	# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
-	# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end	+	# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end

	*For H2B-LOV construct		*For H2B-LOV construct

Vi Nguyen: /* 11/21/12 */

Vi Nguyen — Wed, 05 Dec 2012 23:51:30 GMT

11/21/12

←Older revision		Revision as of 23:51, 5 December 2012
Line 17:		Line 17:
	* Note: Primers add a XbaI site upstream and the SpeI site downstream		* Note: Primers add a XbaI site upstream and the SpeI site downstream
	* Final Gibson assembly products		* Final Gibson assembly products
-	#pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end	+	# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
-	#pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end	+	# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end

	*For H2B-LOV construct		*For H2B-LOV construct

Vi Nguyen: /* 11/21/12 */

Vi Nguyen — Wed, 05 Dec 2012 23:51:16 GMT

11/21/12

←Older revision		Revision as of 23:51, 5 December 2012
Line 17:		Line 17:
	* Note: Primers add a XbaI site upstream and the SpeI site downstream		* Note: Primers add a XbaI site upstream and the SpeI site downstream
	* Final Gibson assembly products		* Final Gibson assembly products
-	pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end	+	#pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
-	pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end	+	#pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end

	*For H2B-LOV construct		*For H2B-LOV construct

Vi Nguyen: /* Entry title */

Vi Nguyen — Wed, 05 Dec 2012 23:49:58 GMT

Entry title

←Older revision		Revision as of 23:49, 5 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 5, 2012==
-	* Insert ~~content here~~...	+	==11/21/12==
		+
		+	'''Results from last week's ligation'''
		+	* None of the colonies on any plates grew
		+	* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
		+	----
		+
		+	* Repeat PCR of H2B and LOV inserts with new primers
		+	* Note: Primers add a XbaI site upstream and the SpeI site downstream
		+	* Final Gibson assembly products
		+	pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
		+	pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
		+
		+	*For H2B-LOV construct
		+	# H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
		+	# LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
		+
		+	*For LOV-H2B construct
		+	# H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
		+	# LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev
		+
		+	Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| Reagents \|\| H2B \|\| LOV
		+	\|-
		+	\| Plasmid DNA \|\| 0.5 μL \|\| 0.5
		+	\|-
		+	\| primer 1 (10 μM) \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| primer 2 (10 μM) \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| 2x GoTaq mix \|\| 12.5 \|\| 12.5
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 10.0 \|\| 10.0
		+	\|-
		+	\| Total \|\| 25.0 \|\| 25.0
		+	\|}
		+
		+	PCR program:
		+	* 95°C, 3 min.
		+	* [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
		+	* 72°C, 3 min.
		+	* 4°C ∞
		+
		+	* Clean up PCR products using the Zymo Clean and Concentrator kit
		+	* Be sure to elute using 15 μL dH<sub>2</sub>O
		+
		+	----
		+
		+	*'''[[User:Karmella Haynes\|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation
		+
		+	'''Assemblies'''
		+	# NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
		+	# NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
		+	# NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
		+	# Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp
		+	# Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp
		+	# Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp
		+
		+	* Digests:
		+	** In a 30 μL reaction, cut H2B with XbaI and SpeI
		+	** In a 30 μL reaction, cut LOV with XbaI and SpeI
		+
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| Reagent \|\| H2B PCR \|\| LOV PCR
		+	\|-
		+	\| DNA \|\| 15.0 \|\| 15.0
		+	\|-
		+	\| XbaI \|\| 1.0\|\| 1.0
		+	\|-
		+	\| SpeI \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| 10x buffer \|\| 3.0 \|\| 3.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 10.0 \|\| 10.0
		+	\|-
		+	\| Total vol. \|\| 30. 0 \|\| 30.0
		+	\|}
		+
		+
		+	* Run entire 30 μL on a 1% gel (use the big fat-tooth well comb)
		+	* Cut band out of gel (use UV box)
		+
		+
		+	Results from gel recovery:
		+	{\| class="wikitable" border=1 cellpadding="5" cellspacing="0"
		+	\|-
		+	\| Sample\|\| 260\|\| 260/280 \|\| ng/μL
		+	\|-
		+	\| H2B - X/S 1 \|\| 0.016 \|\|1.807\|\| 15.961
		+	\|-
		+	\| H2B - X/S 2 \|\| 0.009 \|\| 1.976 \|\| 8.619
		+	\|-
		+	\| LOV - X/S 1 \|\| 0.006\|\| 1.694 \|\| 6.455
		+	\|-
		+	\| LOV - X/S 2 \|\| 0.025\|\| 1.883 \|\| 25.363
		+	\|}
		+
		+
		+	[[Image:VN_Gel_11-21-12.png\|Results of from the digests are shown.]]
		+	[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
		+
		+	# H2B = 410
		+	# LOV = 461
		+
		+	* There were two samples made of each PCR reaction. The two from the H2B are shown on the left, and the two from the LOV are shown on the right.
		+
		+
		+	'''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
		+

Vi Nguyen: Autocreate 2012/12/05 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

Vi Nguyen — Wed, 05 Dec 2012 22:20:41 GMT

Autocreate 2012/12/05 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__