User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09 - Revision history

Karmella Haynes: /* 01/09/13 */

Karmella Haynes — Fri, 11 Jan 2013 00:57:54 GMT

01/09/13

←Older revision		Revision as of 00:57, 11 January 2013
Line 9:		Line 9:
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
	* Golden Gate assembly: hPCD + BL01 + pSB1A3		* Golden Gate assembly: hPCD + BL01 + pSB1A3
		+	* Violacein: plates from Tabor; incubate o/n

Karmella Haynes: /* 01/09/13 */

Karmella Haynes — Thu, 10 Jan 2013 01:48:31 GMT

01/09/13

←Older revision		Revision as of 01:48, 10 January 2013
Line 34:		Line 34:

	* Golden Gate Reactions		* Golden Gate Reactions
		+	# hPCD + BL01 + pSB1A3
		+	# pSB1A3
		+
	{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
	\|- valign="top"		\|- valign="top"
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	\|   \|\| 10.0 \|\| 10.0		\|   \|\| 10.0 \|\| 10.0
	\|}		\|}
		+
		+	Thermal cycler
		+	* [45°C, 2 min.; 16°C, 5 min.] x25
		+	* 60°C, 20 min.
		+	* 80°C, 20 min.

Karmella Haynes: /* 01/09/13 */

Karmella Haynes — Thu, 10 Jan 2013 01:46:39 GMT

01/09/13

←Older revision		Revision as of 01:46, 10 January 2013
Line 16:		Line 16:
	* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites		* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites

-	Dilutions	+	* Dilutions
-	{\| {{table}} border="1" cellspacing="3" <!-- ~~Digest check rxn.~~ table -->	+	{\| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
	\|- valign="top"		\|- valign="top"
	\| bgcolor=#cfcfcf \| DNA		\| bgcolor=#cfcfcf \| DNA
-	\| bgcolor=#cfcfcf \| length	+	\| bgcolor=#cfcfcf \| length (bp)
	\| bgcolor=#cfcfcf \| ng/μL		\| bgcolor=#cfcfcf \| ng/μL
	\| bgcolor=#cfcfcf \| Vol.		\| bgcolor=#cfcfcf \| Vol.
Line 32:		Line 32:
	\|}		\|}

		+
		+	* Golden Gate Reactions
		+	{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
		+	\|- valign="top"
		+	\| Reagent \|\| 1 \|\| 2
		+	\|-
		+	\| gg2 pSB1A3 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| gg3 hPCD \|\| 1.0 \|\| ---
		+	\|-
		+	\| gg4 BL01 \|\| 1.0 \|\| ---
		+	\|-
		+	\| 10x ligase buffer \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| NEB T4 lgase \|\| 0.25 \|\| 0.25
		+	\|-
		+	\| BsmBI \|\| 0.5 \|\| 0.5
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 5.25 \|\| 7.25
		+	\|-
		+	\|   \|\| 10.0 \|\| 10.0
		+	\|}

Karmella Haynes: /* 01/09/13 */

Karmella Haynes — Thu, 10 Jan 2013 01:38:35 GMT

01/09/13

←Older revision		Revision as of 01:38, 10 January 2013
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	\| bgcolor=#cfcfcf \| DNA		\| bgcolor=#cfcfcf \| DNA
	\| bgcolor=#cfcfcf \| length		\| bgcolor=#cfcfcf \| length
-	\| bgcolor=#cfcfcf \| ~~Conc.~~	+	\| bgcolor=#cfcfcf \| ng/μL
	\| bgcolor=#cfcfcf \| Vol.		\| bgcolor=#cfcfcf \| Vol.
	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O		\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O

Karmella Haynes at 01:37, 10 January 2013

Karmella Haynes — Thu, 10 Jan 2013 01:37:58 GMT

←Older revision		Revision as of 01:37, 10 January 2013
Line 20:		Line 20:
	\|- valign="top"		\|- valign="top"
	\| bgcolor=#cfcfcf \| DNA		\| bgcolor=#cfcfcf \| DNA
		+	\| bgcolor=#cfcfcf \| length
	\| bgcolor=#cfcfcf \| Conc.		\| bgcolor=#cfcfcf \| Conc.
	\| bgcolor=#cfcfcf \| Vol.		\| bgcolor=#cfcfcf \| Vol.
	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O		\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O
	\|-		\|-
-	\| gg2 pSB1A3 \|\| 67.7 \|\| \|\|	+	\| gg2 pSB1A3 \|\| 2000 \|\| 67.7 \|\| 7.7 \|\| 12.3
	\|-		\|-
-	\| gg3 hPCD \|\| 22.5 \|\| \|\|	+	\| gg3 hPCD \|\| 186 \|\| 22.5 \|\| 2.1 \|\| 17.8
	\|-		\|-
-	\| gg4 BL01 \|\| 55.9 \|\| \|\|	+	\| gg4 BL01 \|\| 2520 \|\| 55.9 \|\| 11.7 \|\| 8.3
	\|}		\|}

Karmella Haynes: /* 01/09/13 */

Karmella Haynes — Thu, 10 Jan 2013 01:31:10 GMT

01/09/13

←Older revision		Revision as of 01:31, 10 January 2013
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	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O		\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O
	\|-		\|-
-	\| gg2 \|\| 67.7 \|\| \|\|	+	\| gg2 pSB1A3 \|\| 67.7 \|\| \|\|
	\|-		\|-
-	\| gg3 \|\| 22.5 \|\| \|\|	+	\| gg3 hPCD \|\| 22.5 \|\| \|\|
	\|-		\|-
-	\| gg4 \|\| 55.9 \|\| \|\|	+	\| gg4 BL01 \|\| 55.9 \|\| \|\|
	\|}		\|}

Karmella Haynes: /* mm/dd/yy */

Karmella Haynes — Thu, 10 Jan 2013 01:28:42 GMT

mm/dd/yy

←Older revision		Revision as of 01:28, 10 January 2013
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. ##### -->
-	==mm/dd/yy==	+	==01/09/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Golden Gate assembly: hPCD + BL01 + pSB1A3
-	* Line item 2	+


	----		----
-	'''~~Minipreps~~'''<br>	+	'''Golden Gate Assembly'''<br>
-	* ~~Check with E~~/~~P digests~~	+	* Use purified, measured PCR products from 1/05/13
		+	* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites

		+	Dilutions
	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
	\|- valign="top"		\|- valign="top"
-	\| bgcolor=#cfcfcf \| ~~Reagent~~	+	\| bgcolor=#cfcfcf \| DNA
-	\| bgcolor=#cfcfcf \| ~~Volume~~	+	\| bgcolor=#cfcfcf \| Conc.
-	\| ~~rowspan~~=~~"7"~~ \| ~~<u>Expected:</u><br>1~~. ~~BB 1 = size<br>2. BB2 = size<br>~~	+	\| bgcolor=#cfcfcf \| Vol.
-	\| ~~rowspan~~=~~"7"~~ \| <~~!-- [[Image:GelImage.jpg\|400px\|Hover name]]~~<~~br>15 μL~~/~~lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] --~~>	+	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O
	\|-		\|-
-	\| ~~DNA(plasmid)~~ \|\| 2.~~0 μL~~	+	\| gg2 \|\| 67.7 \|\| \|\|
	\|-		\|-
-	\| ~~10X buffer~~ \|\| 1.5	+	\| gg3 \|\| 22.5 \|\| \|\|
	\|-		\|-
-	\| ~~EcoRI~~ \|\| 1.0	+	\| gg4 \|\| 55.9 \|\| \|\|
-	\|-	+
-	~~\| PstI \|\| 1.0~~	+
-	\|-	+
-	~~\| dH<sub>2</sub>O \|\|~~ 9.5	+
-	\|-	+
-	\| ~~ ~~ \|\| ~~15 μL --> 37°C/ ~15 min.~~	+
	\|}		\|}

-	~~----~~
-	~~'''Assemblies'''~~
-	~~# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size~~
-	~~# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size~~
-
-
-	* Digests (Fermentas FD)
-	** Specific notes
-
-	~~{\| {{table}} cellspacing="3" <!-- Digest rxn. table -->~~
-	~~\|- valign="top"~~
-	~~\| bgcolor=#cfcfcf \| Reagent~~
-	~~\| bgcolor=#cfcfcf \| Volume~~
-	~~\| rowspan="7" \| <!-- [[Image:GelImage.jpg\|270px\|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->~~
-	\|-
-	~~\| DNA (plasmid) \|\| up to 25 μL~~
-	\|-
-	~~\| 10x buffer \|\| 3.0~~
-	\|-
-	~~\| enzyme 1 \|\| 1.0~~
-	\|-
-	~~\| enzyme 2 \|\| 1.0~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| ---~~
-	\|-
-	~~\|   \|\| 30 μL --> 37°C/ ~30 min.~~
-	\|}
-
-
-	* Measure conc.'s
-	~~{\| {{table}} cellspacing="3" <!-- [DNA] table -->~~
-	~~\|- bgcolor=#cfcfcf~~
-	~~\| Sample \|\| OD260 \|\| 260/280 \|\| ng/μL~~
-	\|-
-	~~\| 1. Digested part (a/b) \|\| --- \|\| --- \|\| ---~~
-	\|-
-	~~\| 2. Digested part (c/d) \|\| --- \|\| --- \|\| ---~~
-	\|}
-
-
-	* Dephosphorylation (Roche)
-	~~{\| {{table}} cellspacing="3" <!-- Dephos table -->~~
-	\|-
-	~~\| bgcolor=#cfcfcf \| Reagent~~
-	~~\| bgcolor=#cfcfcf \| Volume~~
-	\|-
-	~~\| DNA (clean digest) \|\| up to 17 μL (500 ng)~~
-	\|-
-	~~\| 10x buffer d.p. \|\| 2.0~~
-	\|-
-	~~\| phosphatase \|\| 1.0~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| ---~~
-	\|-
-	~~\|   \|\| 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL~~
-	\|}
-
-
-	* Ligations
-	~~{\| {{table}} cellspacing="3" <!-- Ligations table -->~~
-	~~\|- bgcolor=#cfcfcf~~
-	~~\| Ligation \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>~~
-	\|-
-	~~\| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng \|\| <font color="blue">new BioBrick #:1 (Pick #)</font>~~
-	\|-
-	~~\| 2. vector(c/d)/ ## ng \|\|  ~~
-	\|}
-
-	~~{\| {{table}} cellspacing="3" <!-- Ligation rxn table -->~~
-	~~\|   \|\| 1 \|\| 2 \|\|~~
-	\|-
-	~~\| Insert DNA \|\| ### \|\| --- \|\|~~
-	\|-
-	~~\| Vector DNA \|\| ### \|\| ### \|\|~~
-	\|-
-	~~\| 2x lgn buf (Roche) \|\| ### \|\| ### \|\|~~
-	\|-
-	~~\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\|~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| ### \|\| ### \|\|~~
-	\|-
-	~~\|   \|\| # μL \|\| # μL \|\|~~
-	\|}
-
-	~~----~~
-	~~'''Oligo annealing'''~~
-	~~# New BB 1~~
-	~~# New BB 2~~
-
-	~~{\| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->~~
-	~~\| DNA (oligos, 100 μM) \|\| up to 18 μL (3 μL ea.)~~
-	\|-
-	~~\| 10x annealing buffer \|\| 2.0~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| ---~~
-	\|-
-	~~\|   \|\| 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight~~
-	\|}

Karmella Haynes: Autocreate 2013/01/09 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

Karmella Haynes — Thu, 10 Jan 2013 01:23:29 GMT

Autocreate 2013/01/09 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

New page

{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]Karmella's BioBrick Cloning
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]] {{#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"|

==mm/dd/yy==

* Line item 1
* Line item 2

----
'''Minipreps''' 
* Check with E/P digests

{| {{table}} border="1" cellspacing="3" 
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | Expected: 1. BB 1 = size 2. BB2 = size 
| rowspan="7" | 
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH2O || 9.5
|-
|   || 15 μL --> 37°C/ ~15 min.
|}

----
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size

* Digests (Fermentas FD)
** Specific notes

{| {{table}} cellspacing="3" 
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | 
|-
| DNA (plasmid) || up to 25 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH2O || ---
|-
|   || 30 μL --> 37°C/ ~30 min.
|}

* Measure conc.'s
{| {{table}} cellspacing="3" 
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}

* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" 
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH2O || ---
|-
|   || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}

* Ligations
{| {{table}} cellspacing="3" 
|- bgcolor=#cfcfcf
| Ligation || Plate results (lig : neg crtl) mm/dd/yy
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || new BioBrick #:1 (Pick #)
|-
| 2. vector(c/d)/ ## ng ||  
|}

{| {{table}} cellspacing="3" 
|   || 1 || 2 ||
|-
| Insert DNA || ### || --- ||
|-
| Vector DNA || ### || ### ||
|-
| 2x lgn buf (Roche) || ### || ### ||
|-
| T4 ligase (NEB) || 1.0 || 1.0 ||
|-
| dH2O || ### || ### ||
|-
|   || # μL || # μL ||
|}

----
'''Oligo annealing'''
# New BB 1
# New BB 2

{| class="wikitable" border="0" cellspacing="3" 
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH2O || ---
|-
|   || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}


|}

__NOTOC__

←Older revision		Revision as of 01:38, 10 January 2013
Line 21:		Line 21:
	\| bgcolor=#cfcfcf \| DNA		\| bgcolor=#cfcfcf \| DNA
	\| bgcolor=#cfcfcf \| length		\| bgcolor=#cfcfcf \| length
-	\| bgcolor=#cfcfcf \| ~~Conc.~~	+	\| bgcolor=#cfcfcf \| ng/μL
	\| bgcolor=#cfcfcf \| Vol.		\| bgcolor=#cfcfcf \| Vol.
	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O		\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O

←Older revision		Revision as of 01:37, 10 January 2013
Line 20:		Line 20:
	\|- valign="top"		\|- valign="top"
	\| bgcolor=#cfcfcf \| DNA		\| bgcolor=#cfcfcf \| DNA
		+	\| bgcolor=#cfcfcf \| length
	\| bgcolor=#cfcfcf \| Conc.		\| bgcolor=#cfcfcf \| Conc.
	\| bgcolor=#cfcfcf \| Vol.		\| bgcolor=#cfcfcf \| Vol.
	\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O		\| bgcolor=#cfcfcf \| + dH<sub>2</sub>O
	\|-		\|-
-	\| gg2 pSB1A3 \|\| 67.7 \|\| \|\|	+	\| gg2 pSB1A3 \|\| 2000 \|\| 67.7 \|\| 7.7 \|\| 12.3
	\|-		\|-
-	\| gg3 hPCD \|\| 22.5 \|\| \|\|	+	\| gg3 hPCD \|\| 186 \|\| 22.5 \|\| 2.1 \|\| 17.8
	\|-		\|-
-	\| gg4 BL01 \|\| 55.9 \|\| \|\|	+	\| gg4 BL01 \|\| 2520 \|\| 55.9 \|\| 11.7 \|\| 8.3
	\|}		\|}