User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06 - Revision history

Karmella Haynes: /* 01/06/13 */

Karmella Haynes — Mon, 07 Jan 2013 00:38:13 GMT

01/06/13

←Older revision		Revision as of 00:38, 7 January 2013
Line 61:		Line 61:
	# pSB1A3, 6.0 μL 1/4x Gibson		# pSB1A3, 6.0 μL 1/4x Gibson

-		+	* Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
		+	* Plate on 100 μg/mL amp.

Karmella Haynes: /* 01/06/13 */

Karmella Haynes — Mon, 07 Jan 2013 00:36:59 GMT

01/06/13

←Older revision		Revision as of 00:36, 7 January 2013
Line 30:		Line 30:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| bgcolor=#cfcfcf \| 5x mix		\| bgcolor=#cfcfcf \| 5x mix
		+	\| rowspan="7" \| [[Image:KAH010613_gel1.jpg\|270px\|PCR gel]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| Colony \|\| --- \|\| ---		\| Colony \|\| --- \|\| ---
Line 49:		Line 50:
	* 72°C, 3 min.		* 72°C, 3 min.
	* 4°C, ∞		* 4°C, ∞
		+
		+	Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)


	----		----
-	'''~~Assemblies~~'''	+	'''Transformation'''
-	# ~~BioBrick name: 5' part/(a/b)/size~~ + ~~3' part~~/(c/~~d)/size~~	+	# hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
-	# ~~BioBrick name: 5' part/(a/b)/size~~ + ~~3' part~~/~~(c/d)~~/~~size~~	+	# V0120, 6.0 μL 1/4x Gibson
		+	# hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
		+	# pSB1A3, 6.0 μL 1/4x Gibson


-	* 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: /* 01/06/13 */

Karmella Haynes — Sun, 06 Jan 2013 19:35:11 GMT

01/06/13

←Older revision		Revision as of 19:35, 6 January 2013
Line 8:		Line 8:
	==01/06/13==		==01/06/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* Gibson ~~Asembly~~: pick colonies & check via colony PCR	+	* Gibson Assembly: pick colonies & check via colony PCR
	* Media: Make LB agar +amp		* Media: Make LB agar +amp
		+	* Gibson Assembly: redo transformation with more DNA

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

Karmella Haynes — Sun, 06 Jan 2013 19:20:06 GMT

mm/dd/yy

←Older revision		Revision as of 19:20, 6 January 2013
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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==	+	==01/06/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Gibson Asembly: pick colonies & check via colony PCR
-	* ~~Line item 2~~	+	* Media: Make LB agar +amp


	----		----
-	'''~~Minipreps~~'''<br>	+	'''Gibson Assembly results'''<br>
-	* ~~Check with E~~/~~P digests~~	+	* Plates from [http://openwetware.org/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/05 01/05/2013]

-	{\| {{table}} ~~border="1"~~ cellspacing="3" <!-- ~~Digest check rxn.~~ table -->	+	* hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
-	\|- ~~valign="top"~~	+	* hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
		+	* hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
		+	* hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)
		+
		+	* Pick and streak colonies from plates 3, 5, 7
		+	* Use same pipette tip for colony PCR
		+
		+	{\| {{table}} cellspacing="3" <!-- Dephos table -->
		+	\|-
	\| bgcolor=#cfcfcf \| Reagent		\| bgcolor=#cfcfcf \| Reagent
-	\| bgcolor=#cfcfcf \| Volume	+	\| bgcolor=#cfcfcf \| Volume
-	\| ~~rowspan~~=~~"7"~~ \| ~~<u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>~~	+	\| bgcolor=#cfcfcf \| 5x mix
-	~~\| 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~~	+	\| Colony \|\| --- \|\| ---
	\|-		\|-
-	\| ~~10X buffer~~ \|\| 1.5	+	\| 10 μM primer BL9 \|\| 1.0 \|\| 5.0
	\|-		\|-
-	\| ~~EcoRI~~ \|\| 1.0	+	\| 10 μM primer BL10 \|\| 1.0 \|\| 5.0
	\|-		\|-
-	\| ~~PstI~~ \|\| 1.0	+	\| 2x GoTaq \|\| 12.5 \|\| 62.5
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| 9.5	+	\| dH<sub>2</sub>O \|\| 10.5 \|\| 52.5
	\|-		\|-
-	\|   \|\| ~~15 μL --> 37°C/ ~15 min~~.	+	\|   \|\| 25.0 μL \|\|
	\|}		\|}
		+
		+	PCR
		+	* 95°C, 3 min.
		+	* [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
		+	* 72°C, 3 min.
		+	* 4°C, ∞
		+

	----		----

Karmella Haynes: Autocreate 2013/01/06 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

Karmella Haynes — Sun, 06 Jan 2013 19:04:01 GMT

Autocreate 2013/01/06 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__