IGEM:PennState/Labbook/AudreyLeung - Revision history

ALeung at 19:51, 30 July 2007

2007-07-30T19:51:19Z

←Older revision		Revision as of 19:51, 30 July 2007
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	--Troubleshooting		--Troubleshooting
	*Purification of discarded plates from lab benches may help to increase transformation efficiency		*Purification of discarded plates from lab benches may help to increase transformation efficiency
-	*Try reducing the time of Audrey incubating at room temperature during ligations. This will increase ligation efficiency by 80%

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ALeung: /* Beginning to Build Constructs -- I741015 with E0240 and I741005 with B0015 */

2007-07-23T18:09:39Z

Beginning to Build Constructs -- I741015 with E0240 and I741005 with B0015

←Older revision		Revision as of 18:09, 23 July 2007
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	== Beginning to Build Constructs -- I741015 with E0240 and I741005 with B0015 ==		== Beginning to Build Constructs -- I741015 with E0240 and I741005 with B0015 ==
		+	<br>[[Image:I741015+E0240andI741005+B0015.jpg\|thumb\|Restrictions on Gel]]
	--Did prefix insertion of I741005 into B0015 in pSB1K3		--Did prefix insertion of I741005 into B0015 in pSB1K3
	--Did suffix insertion of E0240 into I741015 in pSB1A2		--Did suffix insertion of E0240 into I741015 in pSB1A2

ALeung at 17:58, 23 July 2007

2007-07-23T17:58:12Z

←Older revision		Revision as of 17:58, 23 July 2007
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	1. To 50 or 100 ul cells, add 5-50 ng DNA. Leave 30’ on ice.		1. To 50 or 100 ul cells, add 5-50 ng DNA. Leave 30’ on ice.
-	2. Heat shock, 45 sec at ~~42C~~, then chill cells on ice about 2’.	+	2. Heat shock, 45 sec at 42°C, then chill cells on ice about 2’.
	3. Spin down (15 sec, eppendorf fuge) and remove SN. (Removing the Manganese		3. Spin down (15 sec, eppendorf fuge) and remove SN. (Removing the Manganese
	seems to boost efficiency about 10X) and resuspend cells in 200 ul LB.		seems to boost efficiency about 10X) and resuspend cells in 200 ul LB.
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	5ul vector (P1010 in pSB1A2)		5ul vector (P1010 in pSB1A2)
	3ul autoclaved, distilled water		3ul autoclaved, distilled water
-	4 hours at ~~37C~~	+	4 hours at 37°C
	*for negative, no enzymes, used water to make up the volume		*for negative, no enzymes, used water to make up the volume

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	<h1>'''July 8, 2007'''</h1>		<h1>'''July 8, 2007'''</h1>
	== Restriction -- P1010 in pSB1A2 ==		== Restriction -- P1010 in pSB1A2 ==
-	--Restricted for 1 hour at ~~37C~~ with EcoRI and SpeI	+	--Restricted for 1 hour at 37°C with EcoRI and SpeI
	--Run all restrictions (including the 20hr one from yesterday) on 1.5% gel		--Run all restrictions (including the 20hr one from yesterday) on 1.5% gel
	*good band for pSB1A2		*good band for pSB1A2
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	--Ligation Experiment 2		--Ligation Experiment 2
	*Using xyl promoter, xylR ORF, xylR total, GFP, pSB1A2		*Using xyl promoter, xylR ORF, xylR total, GFP, pSB1A2
-	*Restrict on DNA already taken from gel after PCR (EcoRI and SpeI) for 3 hours at ~~37C~~ (Restricted 2 hours for GFP and pSB1A2)	+	*Restrict on DNA already taken from gel after PCR (EcoRI and SpeI) for 3 hours at 37°C (Restricted 2 hours for GFP and pSB1A2)
	*Use PCR cleaning kit afterwards (put GFP in freezer after this)		*Use PCR cleaning kit afterwards (put GFP in freezer after this)
	*Ligated with T4 ligase and transformed, using pUC19 as a positive control and no insert for negative control		*Ligated with T4 ligase and transformed, using pUC19 as a positive control and no insert for negative control
-	*Grow O/N at ~~37C~~	+	*Grow O/N at 37°C

	== Ligation and PCR Purification Protocol ==		== Ligation and PCR Purification Protocol ==
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	<br>--Ligation Experiment 2		<br>--Ligation Experiment 2
	*Lots of colonies on xyl promoter (I741015) plate. 20~ish colonies on the xylR ORF (I741005) plate		*Lots of colonies on xyl promoter (I741015) plate. 20~ish colonies on the xylR ORF (I741005) plate
-	*Inoculate I741015 and I741005 into 2ml cultures with Amp overnight ~~37C~~ for miniprepping	+	*Inoculate I741015 and I741005 into 2ml cultures with Amp overnight 37°C for miniprepping
	*xylR total (I741011) plate failed. No colonies		*xylR total (I741011) plate failed. No colonies

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	--Try to digest again from gel for 3 hours, clean with kit (same as Experiment 2 method)		--Try to digest again from gel for 3 hours, clean with kit (same as Experiment 2 method)
	*Used 10ul instead of 12ul of insert with I741011		*Used 10ul instead of 12ul of insert with I741011
-	--Plated and grew overnight at ~~37C~~	+	--Plated and grew overnight at 37°C

	== Preparation for creating constructs with xylR ORF ==		== Preparation for creating constructs with xylR ORF ==

ALeung: /* Gradient PCR Protocol -- PfuUltra DNA polymerase */

2007-07-19T20:56:39Z

Gradient PCR Protocol -- PfuUltra DNA polymerase

←Older revision		Revision as of 20:56, 19 July 2007
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	*50ul 10x PfuUltra HF reaction buffer or ThermolPol buffer (for Taq)		*50ul 10x PfuUltra HF reaction buffer or ThermolPol buffer (for Taq)
	*10ul dNTP (25mM each dNTP)		*10ul dNTP (25mM each dNTP)
-	*0.69 Primer #1	+	*0.69ul Primer #1
-	*0.69 Primer #2	+	*0.69ul Primer #2
	Then aliquoted 39ul mixture into PCR tubes (8 samples, 1 negative) for each polymerase		Then aliquoted 39ul mixture into PCR tubes (8 samples, 1 negative) for each polymerase
	Added 1ul of chromosomal DNA (DNA template) to all tubes except negative control		Added 1ul of chromosomal DNA (DNA template) to all tubes except negative control

ALeung: /* Ligation Troubleshooting */

2007-07-19T14:59:02Z

Ligation Troubleshooting

←Older revision		Revision as of 14:59, 19 July 2007
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	--Single Restrictions using EcoRI and Double Restrictions using SpeI and PstI [http://openwetware.org/wiki/IGEM:PennState/Labbook/NoahJohnson/2007-7-16]		--Single Restrictions using EcoRI and Double Restrictions using SpeI and PstI [http://openwetware.org/wiki/IGEM:PennState/Labbook/NoahJohnson/2007-7-16]
	*double digest don't look good. Only one band seen, not much dropping out. Only 3 of 13 samples appear to have restricted properly.		*double digest don't look good. Only one band seen, not much dropping out. Only 3 of 13 samples appear to have restricted properly.
		+
		+	<h1>July 19, 2007</h1>
		+	http://mycoplasmas.vm.iastate.edu/lab_site/methods/DNA/precip.html

ALeung at 01:04, 19 July 2007

2007-07-19T01:04:19Z

←Older revision		Revision as of 01:04, 19 July 2007
Line 14:		Line 14:
	--Troubleshooting		--Troubleshooting
	*Purification of discarded plates from lab benches may help to increase transformation efficiency		*Purification of discarded plates from lab benches may help to increase transformation efficiency
-	*Try reducing the time of Audrey incubating at room temperature ~~while transformations are resting on ice~~. This will increase ligation efficiency by 80%	+	*Try reducing the time of Audrey incubating at room temperature during ligations. This will increase ligation efficiency by 80%

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ALeung at 01:03, 19 July 2007

2007-07-19T01:03:29Z

←Older revision		Revision as of 01:03, 19 July 2007
Line 14:		Line 14:
	--Troubleshooting		--Troubleshooting
	*Purification of discarded plates from lab benches may help to increase transformation efficiency		*Purification of discarded plates from lab benches may help to increase transformation efficiency
-	*Try reducing the time of Audrey incubating at room temperature while transformations are resting on ice	+	*Try reducing the time of Audrey incubating at room temperature while transformations are resting on ice. This will increase ligation efficiency by 80%

	----		----

ALeung: /* Ligation Troubleshooting */

2007-07-19T01:02:52Z

Ligation Troubleshooting

←Older revision		Revision as of 01:02, 19 July 2007
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	*Try less ligation product for transformation. Anything more than about 5% volume of ligation mix into the chemically competent (Invitrogen) cells started to become inhibitory [http://www.protocol-online.org/biology-forums/posts/14962.html]		*Try less ligation product for transformation. Anything more than about 5% volume of ligation mix into the chemically competent (Invitrogen) cells started to become inhibitory [http://www.protocol-online.org/biology-forums/posts/14962.html]
	*Ligase inhibits transformation. Try denaturing ligase first before transforming		*Ligase inhibits transformation. Try denaturing ligase first before transforming
		+
		+	== Gel ==
		+	--Single Restrictions using EcoRI and Double Restrictions using SpeI and PstI [http://openwetware.org/wiki/IGEM:PennState/Labbook/NoahJohnson/2007-7-16]
		+	*double digest don't look good. Only one band seen, not much dropping out. Only 3 of 13 samples appear to have restricted properly.

ALeung at 00:47, 19 July 2007

2007-07-19T00:47:25Z

←Older revision		Revision as of 00:47, 19 July 2007
Line 14:		Line 14:
	--Troubleshooting		--Troubleshooting
	*Purification of discarded plates from lab benches may help to increase transformation efficiency		*Purification of discarded plates from lab benches may help to increase transformation efficiency
-	*Try reducing the time of Audrey ~~sitting and~~ incubating at room temperature while transformations are resting on ice	+	*Try reducing the time of Audrey incubating at room temperature while transformations are resting on ice

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ALeung at 00:47, 19 July 2007

2007-07-19T00:47:01Z

←Older revision		Revision as of 00:47, 19 July 2007
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	</tr>		</tr>
	</table>		</table>
		+	--Troubleshooting
		+	*Purification of discarded plates from lab benches may help to increase transformation efficiency
		+	*Try reducing the time of Audrey sitting and incubating at room temperature while transformations are resting on ice

	----		----