Janet B. Matsen:Open Lab Questions - Revision history

Janet B. Matsen: /* For biochemical assays, are sodium phosphate and potassium phosphate buffers equivalent? */

2013-05-24T13:28:42Z

For biochemical assays, are sodium phosphate and potassium phosphate buffers equivalent?

←Older revision		Revision as of 13:28, 24 May 2013
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	==Open Questions==		==Open Questions==

-	=== For biochemical assays, are sodium phosphate and potassium phosphate buffers equivalent? ===	+	=== For biochemical assays, are sodium phosphate and potassium phosphate buffers equivalent? 2013/05/24===
-	Are there reasons why it would matter whether sodium or potassium is in solution? I tried to research this question online and haven't found anything. I did learn [[Lidstrom:Buffers#General_Info this]]	+	Are there reasons why it would matter whether sodium or potassium is in solution? I tried to research this question online and haven't found anything. I did learn [[Lidstrom:Buffers#General_Info\|this]]

	=== What is the difference between Bacto yeast extract & Difco yeast extract? ===		=== What is the difference between Bacto yeast extract & Difco yeast extract? ===

Janet B. Matsen: /* Open Questions */

2013-05-24T13:27:59Z

Open Questions

←Older revision		Revision as of 13:27, 24 May 2013
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	==Open Questions==		==Open Questions==
		+
		+	=== For biochemical assays, are sodium phosphate and potassium phosphate buffers equivalent? ===
		+	Are there reasons why it would matter whether sodium or potassium is in solution? I tried to research this question online and haven't found anything. I did learn [[Lidstrom:Buffers#General_Info this]]

	=== What is the difference between Bacto yeast extract & Difco yeast extract? ===		=== What is the difference between Bacto yeast extract & Difco yeast extract? ===

Janet B. Matsen: /* Open Questions */

2013-03-01T22:50:49Z

Open Questions

←Older revision		Revision as of 22:50, 1 March 2013
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	==Open Questions==		==Open Questions==
		+
		+	=== What is the difference between Bacto yeast extract & Difco yeast extract? ===
		+	I e-mailed BD on 2012/03/01 and asked "What is the difference between Difco Yeast Extract and Bacto Yeast Extract? I only see Difco Yeast Extract UF in this: http://www.bd.com/ds/technicalCenter/misc/br_3_2547.pdf so it is hard to compare. I have an old bottle of Difco Yeast extract that is not UF and I wonder if it can be substituted for Bacto Yeast extract. It is also old (1999) -- does it go bad?"
		+
	=== Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) ===		=== Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) ===
	[[image:janet purification gel.jpg\|thumb\|center\|polka-dot DNA in a 0.7% agarose gel]]		[[image:janet purification gel.jpg\|thumb\|center\|polka-dot DNA in a 0.7% agarose gel]]
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	* [http://www.madsci.org/posts/archives/oct2000/971308650.Mi.r.html online]: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.		* [http://www.madsci.org/posts/archives/oct2000/971308650.Mi.r.html online]: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.
	* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.		* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.
-
-

	===How does oxygen limitation cause lower plasmid copy number? What about the growth medium? ===		===How does oxygen limitation cause lower plasmid copy number? What about the growth medium? ===

Janet B. Matsen: /* What causes an overloaded colony PCR reaction to fail? */

2012-11-18T01:45:59Z

What causes an overloaded colony PCR reaction to fail?

←Older revision		Revision as of 01:45, 18 November 2012
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	* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.		* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.

-	~~===What causes an overloaded colony PCR reaction to fail? ===~~	+

	===How does oxygen limitation cause lower plasmid copy number? What about the growth medium? ===		===How does oxygen limitation cause lower plasmid copy number? What about the growth medium? ===

Janet B. Matsen: /* Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) */

2012-11-18T01:04:13Z

Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)

←Older revision		Revision as of 01:04, 18 November 2012
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	===Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) ===		===Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) ===
-	**[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation	+	*[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation
-	** Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."	+	* Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."
-	** [http://www.madsci.org/posts/archives/oct2000/971308650.Mi.r.html online]: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.	+	* [http://www.madsci.org/posts/archives/oct2000/971308650.Mi.r.html online]: ""After making the cells leaky, the DNA is added to the cells and allowed to sit on ice for 10-20 minutes. This allows the DNA to get past the cell membrane, and gives enough time for lots of cells to recieve the DNA and to make certain all of the DNA gets in the cells. After this, in order to make the cells keep the DNA, and to make certain they survive, (Being leaky is not a good thing) the cells are heat shocked for several seconds to 'turn on' (induce) heat shock genes which aid in survival and recovery. After that, the cells are incubated to start growing and plated on selective media to recover those cells that actually recieved the DNA.
-	** It is simple to do an experiment where you transform some without waiting and some after waiting, of course.	+	* It is simple to do an experiment where you transform some without waiting and some after waiting, of course.

	===What causes an overloaded colony PCR reaction to fail? ===		===What causes an overloaded colony PCR reaction to fail? ===

Janet B. Matsen: /* What causes an overloaded colony PCR reaction to fail? */

2012-11-18T01:03:53Z

What causes an overloaded colony PCR reaction to fail?

←Older revision		Revision as of 01:03, 18 November 2012
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	===What causes an overloaded colony PCR reaction to fail? ===		===What causes an overloaded colony PCR reaction to fail? ===

-	*How does plasmid copy number ~~get affected by oxygen availability~~? ~~Cellular media~~?	+	===How does oxygen limitation cause lower plasmid copy number? What about the growth medium? ===
-	**I have noticed that LB often gives ~~me orders of magnitude lower plasmid yields and that growing~~ cultures with liquid:air volumes higher than 1:5 is ~~not good~~.	+	*TB gives ''way'' higher yield than LB
		+	*Growing cultures with liquid:air volumes higher than 1:5 is very bad for plasmid yield.

-	*How does colony density affect colony size? [[image:colony_size_varies_with_colony_density.jpg\|thumb\|center\|colony size varies with colony density]]	+	===Why do apples make people burp? ===
-	**I observe that when there is a large number of colonies per area on an agar plate, that the colonies are smaller. I have thought about the possibility of diffusion-limited arrival of nutrients, however, one would expect to see larger colonies on the outside of the cluster if this is the case.	+	* (posted 5/23/2012)
-		+	** I have been wondering this for years.
-	*Why do apples make people burp? (posted 5/23/2012)	+

Janet B. Matsen: /* Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) */

2012-10-02T17:17:54Z

Why does the DNA clump when you run overloaded agarose gels? (2012/10/02)

←Older revision		Revision as of 17:17, 2 October 2012
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	==Open Questions==		==Open Questions==
	=== Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) ===		=== Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) ===
-	[[image:janet purification gel.jpg\|thumb\|center\|~~include small not~~-~~overloaded lanes when running purification gels~~]]	+	[[image:janet purification gel.jpg\|thumb\|center\|polka-dot DNA in a 0.7% agarose gel]]

	===Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) ===		===Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) ===

Janet B. Matsen: /* Open Questions */

2012-10-02T17:17:30Z

Open Questions

←Older revision		Revision as of 17:17, 2 October 2012
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	==Open Questions==		==Open Questions==
-	*Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)	+	=== Why does the DNA clump when you run overloaded agarose gels? (2012/10/02) ===
		+	[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]
		+
		+	===Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012) ===
	**[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation		**[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation
	** Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."		** Mila weighing in: "This waiting period is part of the protocol. Not sure anyone knows what exactly happens but likely DNA just needs this time to land onto a cell. Longer time does not do much for the efficiency of the transformation. However the efficiency of the cells is determined by something upstream, by how the cells were grown and chemically treated. This is the most important part of the transformation process.."
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	** It is simple to do an experiment where you transform some without waiting and some after waiting, of course.		** It is simple to do an experiment where you transform some without waiting and some after waiting, of course.

-	*What causes an overloaded colony PCR reaction to fail?	+	===What causes an overloaded colony PCR reaction to fail? ===

	*How does plasmid copy number get affected by oxygen availability? Cellular media?		*How does plasmid copy number get affected by oxygen availability? Cellular media?

Janet B. Matsen: /* Open Questions */

2012-08-03T00:55:23Z

Open Questions

←Older revision		Revision as of 00:55, 3 August 2012
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	**I have noticed that LB often gives me orders of magnitude lower plasmid yields and that growing cultures with liquid:air volumes higher than 1:5 is not good.		**I have noticed that LB often gives me orders of magnitude lower plasmid yields and that growing cultures with liquid:air volumes higher than 1:5 is not good.

-	*How does colony density affect colony size? [[image:colony_size_varies_with_colony_density.jpg\|thumb\|colony size varies with colony density]]	+	*How does colony density affect colony size? [[image:colony_size_varies_with_colony_density.jpg\|thumb\|center\|colony size varies with colony density]]
	**I observe that when there is a large number of colonies per area on an agar plate, that the colonies are smaller. I have thought about the possibility of diffusion-limited arrival of nutrients, however, one would expect to see larger colonies on the outside of the cluster if this is the case.		**I observe that when there is a large number of colonies per area on an agar plate, that the colonies are smaller. I have thought about the possibility of diffusion-limited arrival of nutrients, however, one would expect to see larger colonies on the outside of the cluster if this is the case.

	*Why do apples make people burp? (posted 5/23/2012)		*Why do apples make people burp? (posted 5/23/2012)

Janet B. Matsen: /* Open Questions */

2012-07-31T22:22:07Z

Open Questions

←Older revision		Revision as of 22:22, 31 July 2012
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	==Open Questions==		==Open Questions==
-	* Is it ok to re-use electroporation cuvettes after they arc and turn black? (7/31/2012)
-	~~[[image:2012_07 burnt electroporation cuvette.jpg\|thumb\|normal and "burnt" electroporation cuvettes]]~~
-
	*Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)		*Is it important to let chemically competent cells sit on ice 20-30 min after adding DNA? What does this do? (7/13/2012)
	**[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation		**[http://partsregistry.org/Help:Transformation_Protocol\| Example protocol] showing ice incubation