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

2012-12-11T23:38:02Z

mm/dd/yy

←Older revision		Revision as of 23:38, 11 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. ##### -->
-	==mm/dd/yy==	+	==12/10/12==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Retransformation: pSB1A3 (RFP) plasmid from Vi (originally from Rene)
-	* Line item 2	+


	----		----
-	'''~~Minipreps~~'''<br>	+	'''Retransformation'''<br>
-	* Check with E/~~P digests~~	+	# 0.5 μL pSB1A3 (RFP) + 10.0 μL H<sub>2</sub>O
		+	# 10.0 μL H<sub>2</sub>O (neg. ctrl.)

-	~~{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->~~
-	~~\|- valign="top"~~
-	~~\| bgcolor=#cfcfcf \| Reagent~~
-	~~\| bgcolor=#cfcfcf \| Volume~~
-	~~\| 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'''~~
-	~~# 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~~
-	\|}

		+	* Grow for ~8 hours
		+	* Pick two (tiny) colonies; grow in 2 mL LB/Amp overnight
		+	* Return plate to 37 °C (overnight)

	<!-- ##### 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. ##### -->

Karmella Haynes: Autocreate 2012/12/10 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

2012-12-11T23:34:53Z

Autocreate 2012/12/10 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

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{|{{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__

User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/10 - Revision history

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

Karmella Haynes: Autocreate 2012/12/10 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning