User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/17 - Revision history

Karmella Haynes: /* 01/17/13 */

2013-03-03T20:06:28Z

01/17/13

←Older revision		Revision as of 20:06, 3 March 2013
Line 46:		Line 46:
	# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01		# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
	# ggPro-1, VR - correct sequence for SP1AB of BL01		# ggPro-1, VR - correct sequence for SP1AB of BL01
-	# ggPro-2, VF2 - matches ggPro-1 (BLAST)	+	# ggPro-2, VF2 - matches ggPro-1 forward (BLAST)
-	# ggPro-2, VR - matches ggPro-1 (BLAST)	+	# ggPro-2, VR - matches ggPro-1 reverse (BLAST)
-	# ggPro-4, VF2 - matches ggPro-1 (BLAST)	+	# ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
-	#ggPro-4, VR - matches ggPro-1 (BLAST)	+	#ggPro-4, VR - matches ggPro-1 reverse (BLAST)

Karmella Haynes: /* 01/17/13 */

2013-03-03T20:02:14Z

01/17/13

←Older revision		Revision as of 20:02, 3 March 2013
Line 47:		Line 47:
	# ggPro-1, VR - correct sequence for SP1AB of BL01		# ggPro-1, VR - correct sequence for SP1AB of BL01
	# ggPro-2, VF2 - matches ggPro-1 (BLAST)		# ggPro-2, VF2 - matches ggPro-1 (BLAST)
-	# ggPro-1, VR - matches ggPro-1 (BLAST)	+	# ggPro-2, VR - matches ggPro-1 (BLAST)
	# ggPro-4, VF2 - matches ggPro-1 (BLAST)		# ggPro-4, VF2 - matches ggPro-1 (BLAST)
-	#ggPro-1, VR - matches ggPro-1 (BLAST)	+	#ggPro-4, VR - matches ggPro-1 (BLAST)

Karmella Haynes: /* 01/17/13 */

2013-01-24T04:09:41Z

01/17/13

←Older revision		Revision as of 04:09, 24 January 2013
Line 45:		Line 45:
	1/23/13 Sequencing confirmation (DNASU)		1/23/13 Sequencing confirmation (DNASU)
	# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01		# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
-	# ggPro-1, VR -	+	# ggPro-1, VR - correct sequence for SP1AB of BL01
	# ggPro-2, VF2 - matches ggPro-1 (BLAST)		# ggPro-2, VF2 - matches ggPro-1 (BLAST)
-	#	+	# ggPro-1, VR - matches ggPro-1 (BLAST)
	# ggPro-4, VF2 - matches ggPro-1 (BLAST)		# ggPro-4, VF2 - matches ggPro-1 (BLAST)
-	#	+	#ggPro-1, VR - matches ggPro-1 (BLAST)

Karmella Haynes: /* 01/17/13 */

2013-01-24T03:58:56Z

01/17/13

←Older revision		Revision as of 03:58, 24 January 2013
Line 47:		Line 47:
	# ggPro-1, VR -		# ggPro-1, VR -
	# ggPro-2, VF2 - matches ggPro-1 (BLAST)		# ggPro-2, VF2 - matches ggPro-1 (BLAST)
		+	#
		+	# ggPro-4, VF2 - matches ggPro-1 (BLAST)
		+	#

Karmella Haynes: /* 01/17/13 */

2013-01-24T03:57:58Z

01/17/13

←Older revision		Revision as of 03:57, 24 January 2013
Line 41:		Line 41:
	* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background		* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
	* Use BsmBI Type IIS method to assemble all the things!		* Use BsmBI Type IIS method to assemble all the things!
		+
		+
		+	1/23/13 Sequencing confirmation (DNASU)
		+	# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
		+	# ggPro-1, VR -
		+	# ggPro-2, VF2 - matches ggPro-1 (BLAST)

Karmella Haynes: /* 01/17/13 */

2013-01-18T21:24:01Z

01/17/13

←Older revision		Revision as of 21:24, 18 January 2013
Line 37:		Line 37:
	\|}		\|}

-	~~----~~	+	Conclusions
-	'''~~Assemblies~~'''	+	* '''Success!''' (1, 2, & 4) Bands match expected size for vector pSB1A3 (not old vector V0120) and for assembled insert: hPCD (186) + BL01 (2520) = 2706 (visibly larger than BL01 band)
-	~~# BioBrick name: 5' part/~~(~~a/b~~)/size ~~+ 3' part/~~(~~c/d~~)~~/size~~	+	* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
-	~~# BioBrick name~~: ~~5' part/~~(~~a/b~~)~~/size~~ + ~~3' part/~~(~~c/d~~)~~/size~~	+	* Use BsmBI Type IIS method to assemble all the things!

-
-	* 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: /* mm/dd/yy */

2013-01-18T21:16:30Z

mm/dd/yy

←Older revision		Revision as of 21:16, 18 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==	+	==01/17/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Minipreps: check Golden Gate assembly clones (trial 3)
-	* Line item 2	+


Line 15:		Line 15:
	'''Minipreps'''<br>		'''Minipreps'''<br>
	* Check with E/P digests		* Check with E/P digests
		+	* Minipreps from colonies on Plate #2 (Promega home-made ligation buffer trial)

	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 20:		Line 21:
	\| 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>	+	\| rowspan="7" \| <u>Expected:</u><br>1. Plate 2-1 = 2000 (v), 2706 (i)<br>2. Plate 2-2 = 2000 (v), 2706 (i)<br>3. Plate 2-3 (red) = 2000 (v), 2706 (i)<br><br>4. Plate 2-4 = 2000 (v), 2706 (i)<br>5. pSB1A3+RFP = 2000 (v), ~1200 (i)<br>6. hPCD/V0120 = 3200 (v), 186 (i)<br>7. BL01 = 3200 (v), 2520 (i)<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] ~~-->~~	+	\| rowspan="7" \| [[Image:KAH011813_gel1.jpg\|300px\|E/P digests]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
-	\| DNA(plasmid) \|\| 2.0 μL	+	\| DNA(plasmid) \|\| 3.0 μL
	\|-		\|-
	\| 10X buffer \|\| 1.5		\| 10X buffer \|\| 1.5

Karmella Haynes: Autocreate 2013/01/17 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

2013-01-18T21:09:47Z

Autocreate 2013/01/17 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__