Haynes Lab:Notebook/Engineering PC-TFs/2013/01/25 - Revision history

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Fri, 01 Feb 2013 06:52:44 GMT

01/25/13

←Older revision		Revision as of 06:52, 1 February 2013
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	\| bgcolor=#cfcfcf \| ng/μL		\| bgcolor=#cfcfcf \| ng/μL
	\|-		\|-
-	\| 1. pSB1A3 \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 1. pSB1A3 \|\| .084\|\| 1.801 \|\| 83.954
	\|-		\|-
-	\| 2. ~~###~~ \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 2. hPCD \|\| .017 \|\| 1.734\|\| 17.344
	\|-		\|-
-	\| 3. ~~###~~ \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 3. BL02 \|\| .058 \|\| 1.764\|\| 58.413
	\|-		\|-
-	\| 4. ~~###~~ \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 4. BL03 \|\| .082 \|\| 1.831\|\| 81.877
	\|-		\|-
-	\| 5. ~~###~~ \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 5. BL04 \|\| .05 \|\| 1.888 \|\| 49.561
	\|-		\|-
-	\| 6. ~~###~~ \|\| ~~###~~ \|\| ~~###~~ \|\| ~~###~~	+	\| 6. fshPCD \|\| .021 \|\| 1.622 \|\| 21.378
	\|}		\|}

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Fri, 01 Feb 2013 06:37:33 GMT

01/25/13

←Older revision		Revision as of 06:37, 1 February 2013
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	\|-		\|-
	\| 3. ### \|\| ### \|\| ### \|\| ###		\| 3. ### \|\| ### \|\| ### \|\| ###
		+	\|-
		+	\| 4. ### \|\| ### \|\| ### \|\| ###
		+	\|-
		+	\| 5. ### \|\| ### \|\| ### \|\| ###
		+	\|-
		+	\| 6. ### \|\| ### \|\| ### \|\| ###
	\|}		\|}

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Mon, 28 Jan 2013 05:35:58 GMT

01/25/13

←Older revision		Revision as of 05:35, 28 January 2013
Line 51:		Line 51:


-	Purification	+	Purification (Completed on 1-25-2013)
-	* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O	+	* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
-	* Measure [DNA]	+
		+	* Measure [DNA] (1-29-2013)
	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
	\|- valign="top"		\|- valign="top"

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Mon, 28 Jan 2013 05:34:48 GMT

01/25/13

←Older revision		Revision as of 05:34, 28 January 2013
Line 18:		Line 18:

	# pSB1A3, gg0001, gg0002		# pSB1A3, gg0001, gg0002
-	# ~~template~~, ~~primer 1~~, ~~primer 2~~	+	# hPCD, gg0003, gg0004
-	# ~~template~~, ~~primer 1~~, ~~primer 2~~	+	# BL02, gg0005, gg0007
-	# ~~template~~, ~~primer 1~~, ~~primer 2~~	+	# BL03, gg0005, gg0008
		+	# BL04, gg0005, gg0009
		+	# fshPCD, gg0010, gg0012

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Mon, 28 Jan 2013 05:28:33 GMT

01/25/13

←Older revision		Revision as of 05:28, 28 January 2013
Line 27:		Line 27:
	\| bgcolor=#cfcfcf \| Reagent		\| bgcolor=#cfcfcf \| Reagent
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
-	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. ~~BL01~~ = ~~2520~~	+	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br>
	\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]		\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Mon, 28 Jan 2013 05:26:41 GMT

01/25/13

←Older revision		Revision as of 05:26, 28 January 2013
Line 28:		Line 28:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-	\| rowspan="7" \| [[Image:gg_gel1-25-2013\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.2 μL		\| DNA(plasmid) \|\| 0.2 μL

Brady S. Laughlin: /* 01/25/13 */

Brady S. Laughlin — Mon, 28 Jan 2013 05:25:55 GMT

01/25/13

←Older revision		Revision as of 05:25, 28 January 2013
Line 28:		Line 28:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-	\| rowspan="7" \| [[Image:~~image.jpg~~\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:gg_gel1-25-2013\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.2 μL		\| DNA(plasmid) \|\| 0.2 μL

Karmella Haynes: /* 01/25/13 */

Karmella Haynes — Fri, 25 Jan 2013 20:41:16 GMT

01/25/13

←Older revision		Revision as of 20:41, 25 January 2013
Line 43:		Line 43:
	\|}		\|}

-	~~PCR~~
	* 95°C, 3 min.		* 95°C, 3 min.
	* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35		* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
	* 72°C, 3 min.		* 72°C, 3 min.
	* 4°C, ∞		* 4°C, ∞
		+
		+
		+	Purification
		+	* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
		+	* Measure [DNA]
		+	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
		+	\|- valign="top"
		+	\| bgcolor=#cfcfcf \| Sample
		+	\| bgcolor=#cfcfcf \| OD 260
		+	\| bgcolor=#cfcfcf \| 260/280
		+	\| bgcolor=#cfcfcf \| ng/μL
		+	\|-
		+	\| 1. pSB1A3 \|\| ### \|\| ### \|\| ###
		+	\|-
		+	\| 2. ### \|\| ### \|\| ### \|\| ###
		+	\|-
		+	\| 3. ### \|\| ### \|\| ### \|\| ###
		+	\|}

Karmella Haynes: /* 01/25/13 */

Karmella Haynes — Fri, 25 Jan 2013 20:39:54 GMT

01/25/13

←Older revision		Revision as of 20:39, 25 January 2013
Line 17:		Line 17:
	* All of the reactions are as follows:		* All of the reactions are as follows:

-	# ~~template~~, ~~primer 1~~, ~~primer 2~~	+	# pSB1A3, gg0001, gg0002
	# template, primer 1, primer 2		# template, primer 1, primer 2
	# template, primer 1, primer 2		# template, primer 1, primer 2
Line 28:		Line 28:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-	\| rowspan="7" \| [[Image:~~KAH011013_gel1~~.jpg\|200px\|~~Hover name~~]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:image.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.2 μL		\| DNA(plasmid) \|\| 0.2 μL

Karmella Haynes: /* Summary */

Karmella Haynes — Fri, 25 Jan 2013 20:38:02 GMT

Summary

←Older revision		Revision as of 20:38, 25 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. ##### -->
-	==~~Summary~~==	+	==01/25/13==
-	*	+	* Type IIS assembly - day 1
		+
		+
		+	----
		+
		+	PCR
		+	* Resuspend new primers to a final concentration of 100 μM
		+	* Make 10 μM dilutions in separate tubes
		+	* All of the reactions are as follows:
		+
		+	# template, primer 1, primer 2
		+	# template, primer 1, primer 2
		+	# template, primer 1, primer 2
		+	# template, primer 1, primer 2
		+
		+
		+	{\| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table -->
		+	\|- valign="top"
		+	\| bgcolor=#cfcfcf \| Reagent
		+	\| bgcolor=#cfcfcf \| Volume
		+	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
		+	\| rowspan="7" \| [[Image:KAH011013_gel1.jpg\|200px\|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
		+	\|-
		+	\| DNA(plasmid) \|\| 0.2 μL
		+	\|-
		+	\| primer 1 \|\| 1.0
		+	\|-
		+	\| primer 2 \|\| 1.0
		+	\|-
		+	\| 2x GoTaq \|\| 25.0
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 22.8
		+	\|-
		+	\|   \|\| 50.0 μL
		+	\|}
		+
		+	PCR
		+	* 95°C, 3 min.
		+	* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
		+	* 72°C, 3 min.
		+	* 4°C, ∞
		+

←Older revision		Revision as of 05:28, 28 January 2013
Line 27:		Line 27:
	\| bgcolor=#cfcfcf \| Reagent		\| bgcolor=#cfcfcf \| Reagent
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
-	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. ~~BL01~~ = ~~2520~~	+	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL02 = 1431<br> 4. BL03 = 1731<br> 5. BL04 = 1686<br> 6.fshPCD = 186<br>
	\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]		\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-

←Older revision		Revision as of 05:26, 28 January 2013
Line 28:		Line 28:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-	\| rowspan="7" \| [[Image:gg_gel1-25-2013\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:gg_gel1-25-2013.jpg\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.2 μL		\| DNA(plasmid) \|\| 0.2 μL

←Older revision		Revision as of 05:25, 28 January 2013
Line 28:		Line 28:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
-	\| rowspan="7" \| [[Image:~~image.jpg~~\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:gg_gel1-25-2013\|200px\|PCR results]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.2 μL		\| DNA(plasmid) \|\| 0.2 μL