User:Karmella Haynes/Notebook/BioBrick cloning/2012/11/30 - Revision history

Karmella Haynes: /* 11/30/12 */

2013-03-20T03:56:53Z

11/30/12

←Older revision		Revision as of 03:56, 20 March 2013
Line 13:		Line 13:
	----		----
	'''Assemblies'''		'''Assemblies'''
-	# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529	+	# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529

	> Digests (Fermentas FD)<br>		> Digests (Fermentas FD)<br>

Karmella Haynes: /* 11/30/12 */

2012-12-01T21:11:26Z

11/30/12

←Older revision		Revision as of 21:11, 1 December 2012
Line 52:		Line 52:
	\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>		\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
	\|-		\|-
-	\| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng \|\| <font color="blue">KAH3/MV2 #:1 (Pick #)</font>	+	\| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng \|\| <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
	\|-		\|-
-	\| 2. MV2 (S/P)/ 25 ng \|\| ~~ ~~	+	\| 2. MV2 (S/P)/ 25 ng \|\| zero colonies
	\|}		\|}

Karmella Haynes: /* 11/30/12 */

2012-12-01T03:31:14Z

11/30/12

←Older revision		Revision as of 03:31, 1 December 2012
Line 72:		Line 72:
	\|   \|\| 10 μL \|\| 10 μL \|\|		\|   \|\| 10 μL \|\| 10 μL \|\|
	\|}		\|}
		+
		+	* Follow quick transformation protocol
		+	* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar

Karmella Haynes: /* 11/30/12 */

2012-12-01T03:29:24Z

11/30/12

←Older revision		Revision as of 03:29, 1 December 2012
Line 21:		Line 21:
	\|-valign="top"		\|-valign="top"
	\| <u>Reagent</u> \|\| <u>Volume</u> \|\|		\| <u>Reagent</u> \|\| <u>Volume</u> \|\|
-	\| rowspan="9" \| [[Image:~~GelImage~~.~~jpg~~\|~~270px~~\|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="9" \| [[Image:KAH113012_gel1.jpg‎ \|150px\|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		\| DNA (plasmid) \|\| up to 25 μL

Karmella Haynes: /* 11/30/12 */

2012-12-01T02:51:53Z

11/30/12

←Older revision		Revision as of 02:51, 1 December 2012
Line 42:		Line 42:
	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>		\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
	\|-		\|-
-	\| 1. KAH3 (X/P) \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~	+	\| 1. KAH3 (X/P) \|\| 0.028 \|\| 1.96 \|\| 28.41
	\|-		\|-
-	\| 2. MV2 (S/P) \|\| ~~---~~ \|\| ~~---~~ \|\| ~~---~~	+	\| 2. MV2 (S/P) \|\| 0.085 \|\| 1.93 \|\| 85.02
	\|}		\|}

Line 60:		Line 60:
	\|   \|\| 1 \|\| 2 \|\|		\|   \|\| 1 \|\| 2 \|\|
	\|-		\|-
-	\| Insert DNA \|\| ~~###~~ \|\| --- \|\|	+	\| Insert DNA \|\| 0.3 \|\| --- \|\|
	\|-		\|-
-	\| Vector DNA \|\| ~~###~~ \|\| ~~###~~ \|\|	+	\| Vector DNA \|\| 0.3 \|\| 0.3 \|\|
	\|-		\|-
	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\|		\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\|
Line 68:		Line 68:
	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\|		\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\|
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| ~~###~~ \|\| ~~###~~ \|\|	+	\| dH<sub>2</sub>O \|\| 3.4 \|\| 3.7 \|\|
	\|-		\|-
	\|   \|\| 10 μL \|\| 10 μL \|\|		\|   \|\| 10 μL \|\| 10 μL \|\|

Karmella Haynes: /* 11/30/12 */

2012-12-01T01:25:43Z

11/30/12

←Older revision		Revision as of 01:25, 1 December 2012
Line 33:		Line 33:
	\| dH<sub>2</sub>O \|\| ---		\| dH<sub>2</sub>O \|\| ---
	\|-		\|-
-	\|   \|\| 30 μL --> 37°C/ ~30 min.	+	\|   \|\| 30 μL --> 37°C/ ~15 min.
	\|}		\|}

Line 52:		Line 52:
	\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>		\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
	\|-		\|-
-	\| 1. KAH3 (X/P)/751, ## ng + MV2 (S/P)/4529, 25 ng \|\| <font color="blue">KAH3/MV2 #:1 (Pick #)</font>	+	\| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng \|\| <font color="blue">KAH3/MV2 #:1 (Pick #)</font>
	\|-		\|-
	\| 2. MV2 (S/P)/ 25 ng \|\|		\| 2. MV2 (S/P)/ 25 ng \|\|

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

2012-12-01T01:20:27Z

mm/dd/yy

←Older revision		Revision as of 01:20, 1 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. ##### -->
-	==mm/dd/yy==	+	==11/30/12==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment
-	* Line item 2	+

-
-	~~----~~
-	~~'''Minipreps'''<br>~~
-	~~> Check with E/P digests~~
-
-	~~{\| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->~~
-	~~\|-valign="top"~~
-	~~\| <u>Reagent</u> \|\| <u>Volume</u>~~
-	~~\| rowspan="7" \| <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>~~
-	~~\| 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] -->~~
-	\|-
-	~~\| DNA(plasmid) \|\| 2.0 μL~~
-	\|-
-	~~\| 10X buffer \|\| 1.5~~
-	\|-
-	~~\| EcoRI \|\| 1.0~~
-	\|-
-	~~\| PstI \|\| 1.0~~
-	\|-
-	~~\| dH<sub>2</sub>O \|\| 9.5~~
-	\|-
-	~~\|   \|\| 15 μL --> 37°C/ ~15 min.~~
-	\|}

	----		----
	'''Assemblies'''		'''Assemblies'''
-	# ~~BioBrick name~~: ~~5' part/~~(~~a/b~~)~~/size + 3' part~~/(c/d)/~~size~~	+	# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529
-	~~# BioBrick name: 5' part/~~(~~a/b~~)~~/size + 3' part~~/(c/d)/~~size~~	+

	> Digests (Fermentas FD)<br>		> Digests (Fermentas FD)<br>
-	~~--> Specific notes<br>~~	+

	{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
	\|-valign="top"		\|-valign="top"
	\| <u>Reagent</u> \|\| <u>Volume</u> \|\|		\| <u>Reagent</u> \|\| <u>Volume</u> \|\|
-	\| rowspan="9" \| ~~<!--~~ [[Image:GelImage.jpg\|270px\|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] ~~-->~~	+	\| rowspan="9" \| [[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		\| DNA (plasmid) \|\| up to 25 μL
Line 62:		Line 37:


-	> Measure conc.'s	+	> Measure conc.'s of gel purified fragments
	{\| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
	\|-		\|-
	\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>		\| <u>Sample</u> \|\| <u>OD260</u> \|\| <u>260/280</u> \|\| <u>ng/μL</u>
	\|-		\|-
-	\| 1. ~~Digested part~~ (a/b) \|\| --- \|\| --- \|\| ---	+	\| 1. KAH3 (X/P) \|\| --- \|\| --- \|\| ---
	\|-		\|-
-	\| 2. ~~Digested part~~ (c/d) \|\| --- \|\| --- \|\| ---	+	\| 2. MV2 (S/P) \|\| --- \|\| --- \|\| ---
-	\|}	+
-		+
-		+
-	~~> Dephosphorylation (Roche)~~	+
-	~~{\| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->~~	+
-	\|-	+
-	~~\| <u>Reagent</u> \|\| <u>Volume</u>~~	+
-	\|-	+
-	~~\| 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~~	+
	\|}		\|}

Line 92:		Line 50:
	> Ligations		> Ligations
	{\| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->		{\| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
-	\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>	+	\| <u>Ligation</u> \|\| <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
	\|-		\|-
-	\| 1. ~~insert~~(a/b)/~~size~~, ## ng + ~~vector~~(c/d)/~~size~~, ## ng \|\| <font color="blue">~~new BioBrick~~ #:1 (Pick #)</font>	+	\| 1. KAH3 (X/P)/751, ## ng + MV2 (S/P)/4529, 25 ng \|\| <font color="blue">KAH3/MV2 #:1 (Pick #)</font>
	\|-		\|-
-	\| 2. ~~vector~~(c/d)/ ## ng \|\|	+	\| 2. MV2 (S/P)/ 25 ng \|\|
	\|}		\|}

Line 106:		Line 64:
	\| Vector DNA \|\| ### \|\| ### \|\|		\| Vector DNA \|\| ### \|\| ### \|\|
	\|-		\|-
-	\| 2x lgn buf (Roche) \|\| ~~###~~ \|\| ~~###~~ \|\|	+	\| 2x lgn buf (Roche) \|\| 5.0 \|\| 5.0 \|\|
	\|-		\|-
	\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\|		\| T4 ligase (NEB) \|\| 1.0 \|\| 1.0 \|\|
Line 112:		Line 70:
	\| dH<sub>2</sub>O \|\| ### \|\| ### \|\|		\| dH<sub>2</sub>O \|\| ### \|\| ### \|\|
	\|-		\|-
-	\|   \|\| # μL \|\| # μL \|\|	+	\|   \|\| 10 μL \|\| 10 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 2012/11/30 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

2012-12-01T00:49:10Z

Autocreate 2012/11/30 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

{| class="wikitable" border="0" cellspacing="3" 
|-valign="top"
| Reagent || 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 

{| class="wikitable" border="0" cellspacing="3" 
|-valign="top"
| Reagent || Volume ||  
| rowspan="9" | 
|-
| 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
{| class="wikitable" border="0" cellspacing="3" 
|-
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}

> Dephosphorylation (Roche)
{| class="wikitable" border="0" cellspacing="3" 
|-
| Reagent || 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
{| class="wikitable" border="0" cellspacing="3" 
| 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 ||  
|}

{| class="wikitable" border="0" 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__