User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05 - Revision history

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 02:42:11 GMT

01/05/13

←Older revision		Revision as of 02:42, 6 January 2013
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	==01/05/13==		==01/05/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* Gibson~~: PCR parts; waiting on BsmBI from NEB~~	+	* Gibson: PCR parts, complete protocol for Brady's construct
-	* Golden gate: PCR parts, complete protocol for Brady's construct	+	* Golden gate: PCR parts; waiting on BsmBI from NEB

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 02:41:47 GMT

01/05/13

←Older revision		Revision as of 02:41, 6 January 2013
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	==01/05/13==		==01/05/13==
	<!-- Precede finished items with a checkmark ✓ -->		<!-- Precede finished items with a checkmark ✓ -->
-	* Golden gate: PCR parts, complete protocol for Brady's construct
	* Gibson: PCR parts; waiting on BsmBI from NEB		* Gibson: PCR parts; waiting on BsmBI from NEB
		+	* Golden gate: PCR parts, complete protocol for Brady's construct

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 02:40:00 GMT

01/05/13

←Older revision		Revision as of 02:40, 6 January 2013
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	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520
-	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|~~Hover name~~]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.5 μL		\| DNA(plasmid) \|\| 0.5 μL
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	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520
-	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|~~Hover name~~]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.5 μL		\| DNA(plasmid) \|\| 0.5 μL

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 02:39:06 GMT

01/05/13

←Older revision		Revision as of 02:39, 6 January 2013
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	* 4°C, ∞		* 4°C, ∞

-	'''~~Measure~~ DNA Concentrations'''	+
		+	'''Purification'''
		+	* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
		+	* Eluted samples with 20 μL dH<sub>2</sub>O
		+
		+
		+	'''DNA Concentrations'''
	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
	\|- valign="top"		\|- valign="top"
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-	'''~~Measure~~ DNA Concentrations'''	+
		+	'''Purification'''
		+	* Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
		+	* Eluted samples with 20 μL dH<sub>2</sub>O
		+
		+
		+	'''DNA Concentrations'''
	{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->		{\| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
	\|- valign="top"		\|- valign="top"

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 02:18:13 GMT

01/05/13

←Older revision		Revision as of 02:18, 6 January 2013
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	'''Transformation'''		'''Transformation'''
-	* (1) Transform: add '''2.0 μL''' to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar	+	* (1) Transform: add '''2.0 μL''' to 40 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
	* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA, [http://www.neb.com/nebecomm/products/protocol820.asp]		* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA, [http://www.neb.com/nebecomm/products/protocol820.asp]
	* Test both methods (8 plates total)		* Test both methods (8 plates total)

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 01:07:24 GMT

01/05/13

←Older revision		Revision as of 01:07, 6 January 2013
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	# hPCD + BL01 + pSB1A3		# hPCD + BL01 + pSB1A3

-	* Check the sequences for BsmBI sites!	+
		+	* Check the sequences for BsmBI docking sites (CGTCTC)
		+	** hPCD - none
		+	** BL01: hPCD-mCherry-SP1AB - none
		+	** V0120 - <font color="red">5 sites</font>
		+	** pSB1A3 - none
		+

	'''PCR'''		'''PCR'''

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 00:52:12 GMT

01/05/13

←Older revision		Revision as of 00:52, 6 January 2013
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	'''Transformation'''		'''Transformation'''
	* (1) Transform: add '''2.0 μL''' to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar		* (1) Transform: add '''2.0 μL''' to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
-	* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA	+	* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA, [http://www.neb.com/nebecomm/products/protocol820.asp]
	* Test both methods (8 plates total)		* Test both methods (8 plates total)
	** Plates 1-4, method 1		** Plates 1-4, method 1

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 00:45:14 GMT

01/05/13

←Older revision		Revision as of 00:45, 6 January 2013
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	\| dH<sub>2</sub>O \|\| --- \|\| 3.6 \|\| --- \|\| 3.7		\| dH<sub>2</sub>O \|\| --- \|\| 3.6 \|\| --- \|\| 3.7
	\|-		\|-
-	\|   \|\| 5.0 μL	+	\|   \|\| 5.0 μL \|\| 5.0 μL \|\| 5.0 μL \|\| 5.0 μL
	\|}		\|}

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 00:44:44 GMT

01/05/13

←Older revision		Revision as of 00:44, 6 January 2013
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	* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL		* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
	* Thermal cycler: 50°C,60 min.; 4°C, ∞		* Thermal cycler: 50°C,60 min.; 4°C, ∞
-	* Transform: add 20 μL to 50 μL DH5α-Turbo; ice/5 min.; plate on 100 μg/mL amp.	+
		+
		+	'''Transformation'''
		+	* (1) Transform: add '''2.0 μL''' to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
		+	* (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH<sub>2</sub>O (20.0 total vol.), and using 2.0 μL diluted DNA
		+	* Test both methods (8 plates total)
		+	** Plates 1-4, method 1
		+	** Plates 5-8, method 2

Karmella Haynes: /* 01/05/13 */

Karmella Haynes — Sun, 06 Jan 2013 00:35:58 GMT

01/05/13

←Older revision		Revision as of 00:35, 6 January 2013
Line 68:		Line 68:

	'''Reaction set-up'''		'''Reaction set-up'''
-	* Use 0.2 - 0.5 pmol, per NEB's suggestion	+	* Use 0.2 - 0.5 pmol, per NEB's suggestion, [http://www.neb.com/nebecomm/products/protocol819.asp]
	* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000		* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000
	* Total volume of DNA = 5.0 μL max		* Total volume of DNA = 5.0 μL max
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	* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL		* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
	* Thermal cycler: 50°C,60 min.; 4°C, ∞		* Thermal cycler: 50°C,60 min.; 4°C, ∞
-	* Transform	+	* Transform: add 20 μL to 50 μL DH5α-Turbo; ice/5 min.; plate on 100 μg/mL amp.

←Older revision		Revision as of 02:40, 6 January 2013
Line 29:		Line 29:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>G1. V0120 = 3200<br>G2. pSB1A3 = 2000<br>G3. hPCD = 186<br>G4. BL01 = 2520
-	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|~~Hover name~~]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.5 μL		\| DNA(plasmid) \|\| 0.5 μL
Line 148:		Line 148:
	\| bgcolor=#cfcfcf \| Volume		\| bgcolor=#cfcfcf \| Volume
	\| rowspan="7" \| <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520		\| rowspan="7" \| <u>Expected:</u><br>gg1. V0120 = 3200<br>gg2. pSB1A3 = 2000<br>gg3. hPCD = 186<br>gg4. BL01 = 2520
-	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|~~Hover name~~]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]	+	\| rowspan="7" \| [[Image:KAH010513_gel1.jpg\|300px\|PCR gel]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
	\|-		\|-
	\| DNA(plasmid) \|\| 0.5 μL		\| DNA(plasmid) \|\| 0.5 μL