Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/26 - Revision history

Vi Nguyen: /* November 26, 2012 */

2012-12-09T00:41:36Z

November 26, 2012

Vi Nguyen: /* November 26, 2012 */

2012-12-09T00:39:59Z

November 26, 2012

Vi Nguyen: /* November 26, 2012 */

2012-12-09T00:37:38Z

November 26, 2012

←Older revision		Revision as of 00:37, 9 December 2012
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	==November 26, 2012==		==November 26, 2012==
-	* ~~Waiting for~~ Gibson assembly primers to ~~arrive~~	+	'''Gibson assembly written by Dr. Haynes'''
		+	*All 8 primers were ordered
		+
		+	The Gibson assembly method joins double stranded fragments that have overlapping matching ends. Matching ends (instead of BioBrick cut sites) are artificially added via PCR. To do Gibson assembly, we need primers that span 40 bp across junctions of the individual parts.
		+
		+
		+
		+
		+
		+	We will cut the vector with XbaI and SpeI so the ends will look like this:
		+
		+	pSB1A3 left end: …tctggaattcgcggccgctt/
		+
		+	pSB1A3 right end: /ctagtagcggccgctgcagt…
		+
		+
		+
		+
		+	We want to join the following, and add a "6-histidine" tag (and stop codon) for antibody staining:
		+
		+	1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
		+
		+	…to get a circular plasmid.
		+
		+
		+
		+
		+	Let's also try swapping LOV and H2B (like you suggested before) just to see what happens
		+
		+	2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
		+
		+
		+
		+
		+	Each part gets a 40 bp forward and reverse primer. A dash indicates the junction between the parts:
		+
		+
		+
		+
		+	For assembly 1, amplify H2B with
		+	1."1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC
		+	2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG
		+
		+	…and amplify LOV with
		+	1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG
		+	2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT
		+
		+	After PCR of each part, the two PCR products will be mixed with the X/S cut vector and subjected to isothermal assembly: http://openwetware.org/wiki/Haynes:Gibson_Assembly
		+
		+
		+
		+
		+	For assembly 2, amplify LOV with
		+	1."1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG
		+	2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT
		+
		+	…and amplify H2B with
		+	1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC
		+	2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG
		+
		+
		+

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Vi Nguyen: /* Entry title */

2012-12-08T13:40:04Z

Entry title

←Older revision		Revision as of 13:40, 8 December 2012
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-	==~~Entry title~~==	+	==November 26, 2012==
-	* ~~Insert content here...~~	+	* Waiting for Gibson assembly primers to arrive
-		+

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Vi Nguyen: Autocreate 2012/11/26 Entry for Haynes_Lab:Notebook/Investigating_Photo-Switchable_Synthetic_Nucleosomes

2012-12-08T13:39:39Z

Autocreate 2012/11/26 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>}}
|-
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==Entry title==
* Insert content here...


|}

__NOTOC__

←Older revision		Revision as of 00:41, 9 December 2012
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-	For assembly 1, amplify H2B with	+	*For assembly 1, amplify H2B with
-	1."1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC	+	# "1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC
-	2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG	+	# "H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG

-	…and amplify LOV with	+	*…and amplify LOV with
-	1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG	+	# "H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG
-	2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT	+	# "LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT

	After PCR of each part, the two PCR products will be mixed with the X/S cut vector and subjected to isothermal assembly: http://openwetware.org/wiki/Haynes:Gibson_Assembly		After PCR of each part, the two PCR products will be mixed with the X/S cut vector and subjected to isothermal assembly: http://openwetware.org/wiki/Haynes:Gibson_Assembly
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-	For assembly 2, amplify LOV with	+	* For assembly 2, amplify LOV with
-	1."1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG	+	# "1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG
-	2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT	+	# "LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT

-	…and amplify H2B with	+	* …and amplify H2B with
-	1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC	+	# "LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC
-	2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG	+	# "H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG

←Older revision		Revision as of 00:39, 9 December 2012
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	For assembly 1, amplify H2B with		For assembly 1, amplify H2B with
-	1."1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC	+	1."1A3/H2B fwd", 5'- tctggaattcgcggccgcttCTAGA-ATGCCAGAGCCAGCGAAGTC
	2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG		2."H2B/LOV rev", 5'-CGTTCAAGTGTAGTAGCCAA-CTTAGCGCTGGTGTACTTGG

	…and amplify LOV with		…and amplify LOV with
-	1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG	+	1."H2B/LOV fwd", 5'-CCAAGTACACCAGCGCTAAG-TTGGCTACTACACTTGAACG
	2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT		2."LOV+his/1A3 rev", 5'- actgcagcggccgctactagT-ttagtggtgatggtgatgatg-AAGTTCTTTTGCCGCCTCAT

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	For assembly 2, amplify LOV with		For assembly 2, amplify LOV with
-	1."1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG	+	1."1A3/LOV fwd", 5'-tctggaattcgcggccgcttCTAGA-TTGGCTACTACACTTGAACG
	2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT		2."LOV/H2B rev", 5'-GACTTCGCTGGCTCTGGCAT-AAGTTCTTTTGCCGCCTCAT

	…and amplify H2B with		…and amplify H2B with
-	1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC	+	1."LOV/H2B fwd", 5'-ATGAGGCGGCAAAAGAACTT-ATGCCAGAGCCAGCGAAGTC
	2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG		2."H2B+his/1A3 rev", 5'-actgcagcggccgctactagT-ttagtggtgatggtgatgatg-CTTAGCGCTGGTGTACTTGG