User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15 - Revision history

Karmella Haynes at 22:02, 17 January 2013

Karmella Haynes — Thu, 17 Jan 2013 22:02:01 GMT

←Older revision		Revision as of 22:02, 17 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. ##### -->
		+
	==01/15/13==		==01/15/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Thu, 17 Jan 2013 01:12:51 GMT

01/15/13

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Thu, 17 Jan 2013 01:11:03 GMT

01/15/13

←Older revision		Revision as of 01:11, 17 January 2013
Line 37:		Line 37:
	9, 10. Roche - hPCD+BL01+pSB1A3<br>		9, 10. Roche - hPCD+BL01+pSB1A3<br>
	11, 12. Roche - pSB1A3<br>		11, 12. Roche - pSB1A3<br>
-
-
	{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
	\|- valign="top"		\|- valign="top"
Line 64:		Line 62:
	* 60°C, 20 min.		* 60°C, 20 min.
	* 80°C, 20 min.		* 80°C, 20 min.
		+	* 4°C, ∞
		+
		+
		+	* Transformation
		+	** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar
		+	** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Thu, 17 Jan 2013 01:07:08 GMT

01/15/13

←Older revision		Revision as of 01:07, 17 January 2013
Line 47:		Line 47:
	\| gg3 hPCD \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---		\| gg3 hPCD \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---
	\|-		\|-
-	\| gg4 BL01 \|\| 1.0 \|\| ---	+	\| gg4 BL01 \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---
	\|-		\|-
-	\| 10x ligase buffer \|\| 1.0 \|\| 1.0	+	\| 10x ligase buffer \|\| 1.0 \|\| 1.0\|\| 1.0 \|\| 1.0\|\| 5.0 \|\| 5.0
	\|-		\|-
-	\| NEB T4 lgase \|\| 0.25 \|\| 0.25	+	\| NEB T4 lgase \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25
	\|-		\|-
-	\| BsmBI \|\| 0.5 \|\| 0.5	+	\| BsmBI \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5
	\|-		\|-
-	\| dH<sub>2</sub>O \|\| 5.25 \|\| 7.25	+	\| dH<sub>2</sub>O \|\| 5.25 \|\| 7.25 \|\| 5.25 \|\| 7.25 \|\| 1.25 \|\| 3.25
	\|-		\|-
-	\|   \|\| 10.0 \|\| 10.0	+	\|   \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0
	\|}		\|}

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Thu, 17 Jan 2013 00:46:19 GMT

01/15/13

←Older revision		Revision as of 00:46, 17 January 2013
Line 27:		Line 27:
	\|}		\|}

-	* Make Promega buffer from scratch. ~~Test~~ Promega ~~and~~ NEB ~~formulas~~.	+	* Make Promega buffer from scratch.
-	* ~~Follow same procedure as ###~~	+
-	* ~~Transform into ### cells~~	+
		+	* Golden Gate assembly reactions
		+	1, 2. Promega - hPCD+BL01+pSB1A3<br>
		+	3, 4. Promega - pSB1A3<br>
		+	5, 6. NEB - hPCD+BL01+pSB1A3<br>
		+	7, 8. NEB - pSB1A3<br>
		+	9, 10. Roche - hPCD+BL01+pSB1A3<br>
		+	11, 12. Roche - pSB1A3<br>
		+
		+
		+	{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
		+	\|- valign="top"
		+	\| Reagent \|\| Promega (1,2) \|\| Promega (3, 4) \|\| NEB (5, 6) \|\| NEB (7, 8) \|\| Roche (9, 10) \|\| Roche (11, 12)
		+	\|-
		+	\| gg2 pSB1A3 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| gg3 hPCD \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 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
		+	\|}
		+
		+	Thermal cycler
		+	* [45°C, 2 min.; 16°C, 5 min.] x25
		+	* 60°C, 20 min.
		+	* 80°C, 20 min.
		+
		+

	----		----

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Tue, 15 Jan 2013 22:49:43 GMT

01/15/13

←Older revision		Revision as of 22:49, 15 January 2013
Line 28:		Line 28:

	* Make Promega buffer from scratch. Test Promega and NEB formulas.		* Make Promega buffer from scratch. Test Promega and NEB formulas.
		+	* Follow same procedure as ###
		+	* Transform into ### cells

	----		----

Karmella Haynes: /* 01/15/13 */

Karmella Haynes — Tue, 15 Jan 2013 22:48:38 GMT

01/15/13

←Older revision		Revision as of 22:48, 15 January 2013
Line 27:		Line 27:
	\|}		\|}

-	~~----~~	+	* Make Promega buffer from scratch. Test Promega and NEB formulas.
-	~~'''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~~
-	\|}
-

	<!-- ##### 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: /* 01/15/13 */

Karmella Haynes — Tue, 15 Jan 2013 22:46:44 GMT

01/15/13

←Older revision		Revision as of 22:46, 15 January 2013
Line 24:		Line 24:
	\| 10x NEB \|\| 500 mM (pH 7.5) \|\| 100 mM \|\| 100 mM \|\| 10 mM		\| 10x NEB \|\| 500 mM (pH 7.5) \|\| 100 mM \|\| 100 mM \|\| 10 mM
	\|-		\|-
-	\| 2x Roche ~~\|\| ?~~ \|\| ? \|\| ? \|\| ? \|\| ?	+	\| 2x Roche \|\| ? \|\| ? \|\| ? \|\| ?
	\|}		\|}

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

Karmella Haynes — Tue, 15 Jan 2013 22:46:35 GMT

mm/dd/yy

←Older revision		Revision as of 22:46, 15 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==	+	==01/15/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* ~~Line item 1~~	+	* Golden gate assembly: test different ligase buffers
-	* Line item 2	+


	----		----
-	'''~~Minipreps~~'''<br>	+	'''Golden Gate Assembly optimization'''<br>
-	* ~~Check with E/P digests~~	+	* Test different buffers

	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
-	\|- valign="top"	+	\|- valign="top" bgcolor=#cfcfcf
-	\| bgcolor=#cfcfcf ~~\| Reagent~~	+	\| Buffer \|\| Tris-HCl \|\| MgCl<sub>2</sub> \|\| DTT \|\| ATP
-	\| ~~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	+	\| 10x Promega \|\| 300 mM (pH 7.8) \|\| 100 mM \|\| 100 mM \|\| 10 mM
	\|-		\|-
-	\| ~~dH<sub>2</sub>O~~ \|\| 9.5	+	\| 10x NEB \|\| 500 mM (pH 7.5) \|\| 100 mM \|\| 100 mM \|\| 10 mM
	\|-		\|-
-	\| ~~ ~~ \|\| ~~15 μL --> 37°C/ ~15 min.~~	+	\| 2x Roche \|\| ? \|\| ? \|\| ? \|\| ? \|\| ?
	\|}		\|}

Karmella Haynes: Autocreate 2013/01/15 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning

Karmella Haynes — Tue, 15 Jan 2013 22:38:54 GMT

Autocreate 2013/01/15 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 01:12, 17 January 2013
Line 41:		Line 41:
	\| Reagent \|\| Promega (1,2) \|\| Promega (3, 4) \|\| NEB (5, 6) \|\| NEB (7, 8) \|\| Roche (9, 10) \|\| Roche (11, 12)		\| Reagent \|\| Promega (1,2) \|\| Promega (3, 4) \|\| NEB (5, 6) \|\| NEB (7, 8) \|\| Roche (9, 10) \|\| Roche (11, 12)
	\|-		\|-
-	\| gg2 pSB1A3 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0	+	\| gg2 pSB1A3* \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0 \|\| 1.0
	\|-		\|-
-	\| gg3 hPCD \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---	+	\| gg3 hPCD* \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---
	\|-		\|-
-	\| gg4 BL01 \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---	+	\| gg4 BL01* \|\| 1.0 \|\| --- \|\| 1.0 \|\| --- \|\| 1.0 \|\| ---
	\|-		\|-
-	\| ~~10x~~ ligase buffer \|\| 1.0 \|\| 1.0\|\| 1.0 \|\| 1.0\|\| 5.0 \|\| 5.0	+	\| ligase buffer \|\| 1.0 \|\| 1.0\|\| 1.0 \|\| 1.0\|\| 5.0 \|\| 5.0
	\|-		\|-
-	\| NEB T4 ~~lgase~~ \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25	+	\| NEB T4 ligase \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25 \|\| 0.25
	\|-		\|-
-	\| BsmBI \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5	+	\| NEB BsmBI \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5 \|\| 0.5
	\|-		\|-
	\| dH<sub>2</sub>O \|\| 5.25 \|\| 7.25 \|\| 5.25 \|\| 7.25 \|\| 1.25 \|\| 3.25		\| dH<sub>2</sub>O \|\| 5.25 \|\| 7.25 \|\| 5.25 \|\| 7.25 \|\| 1.25 \|\| 3.25
Line 57:		Line 57:
	\|   \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0		\|   \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0 \|\| 10.0
	\|}		\|}
		+	Note: *DNA is 20 fmole/μL

	Thermal cycler		Thermal cycler
Line 65:		Line 66:


-	* ~~Transformation~~	+	* Transformations
	** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar		** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar
	** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar		** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar