Janet B. Matsen:Closed Lab Questions - Revision history

Janet B. Matsen: /* How does the density of electrocompetent cells affect transformation efficiency? */

2013-02-21T18:21:41Z

How does the density of electrocompetent cells affect transformation efficiency?

←Older revision		Revision as of 18:21, 21 February 2013
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	=== How does the density of electrocompetent cells affect transformation efficiency?===		=== How does the density of electrocompetent cells affect transformation efficiency?===
	[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]		[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
-	* It matters a LOT! The undiluted samples (dilution factor = 0) were harvested ad mid-exponential phase and resuspended in 1/500<sup>ths</sup> of the culture volume. Details of ~~the~~ experiment are [https://docs.google.com/document/d/1btKWgYd1GemMa2mBmg-U72bnu2knSyjf_yGqXo8P1nY/edit here].	+	* It matters a LOT! The undiluted samples (dilution factor = 0) were harvested ad mid-exponential phase and resuspended in 1/500<sup>ths</sup> of the culture volume. Details of my experiment are [https://docs.google.com/document/d/1btKWgYd1GemMa2mBmg-U72bnu2knSyjf_yGqXo8P1nY/edit here].

	=== What is the ballpark efficiency of electroporation? ===		=== What is the ballpark efficiency of electroporation? ===

Janet B. Matsen: /* Why do we grow E. coli on glycerol, not glucose? */

2013-02-21T18:21:13Z

Why do we grow E. coli on glycerol, not glucose?

←Older revision		Revision as of 18:21, 21 February 2013
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	=== Why do we grow E. coli on glycerol, not glucose? ===		=== Why do we grow E. coli on glycerol, not glucose? ===
-	* Amanda Smith found:	+	* [[User:Amanda_L_Smith\|Amanda Smith]] found:
	**"Glycerol play the part of a cheap carbon-energy source for E.coli.		**"Glycerol play the part of a cheap carbon-energy source for E.coli.
	*** Ecoli grown on glycerol excretes less acetate compared to e coli grown on glucose. (due to changes in the biochemical pathways and slower absorption rate of glycerol). Acetate is bad because it limits the growth of e coli. This is due primarily by methionine synthesis inhibition, which in turn disrupts protein synthesis and alters protein expression (A horror story if you are expressing proteins). Acetate also acidifies the media which also cause other changes in E coli physiology (such as reduced growth rate, altered K/Na ion and amino acid anions concentration. Which also has effects on protein synthesis)"		*** Ecoli grown on glycerol excretes less acetate compared to e coli grown on glucose. (due to changes in the biochemical pathways and slower absorption rate of glycerol). Acetate is bad because it limits the growth of e coli. This is due primarily by methionine synthesis inhibition, which in turn disrupts protein synthesis and alters protein expression (A horror story if you are expressing proteins). Acetate also acidifies the media which also cause other changes in E coli physiology (such as reduced growth rate, altered K/Na ion and amino acid anions concentration. Which also has effects on protein synthesis)"

Janet B. Matsen: /* Lab Basics: */

2013-02-21T18:20:09Z

Lab Basics:

←Older revision		Revision as of 18:20, 21 February 2013
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	* 70% is still concentrated enough to be very flamable!		* 70% is still concentrated enough to be very flamable!

-	~~===Wow.. these 2 batches of TB media are very different in color... did I mess up?===~~
-	*A:Maybe, but not necessarily. Different batches of yeast and tryptone can have very different compositions. This is one of the purposes of defined media. [[Image:Two different TB batches.jpg\|thumb\|these two TB batches were made with the same recipe but different bottles of one of the yeast-derived reagents. I forget whether it was yeast extract or tryptone.]]
	===How does colony size vary with colony density? ===		===How does colony size vary with colony density? ===
	* As the number of colonies per unit area increases, nutrient diffusion becomes limiting. New molecular biologists are sometimes worried that variation in colony sizes on a plate indicates contamination, but it can be caused by tight packing. [[image:colony_size_varies_with_colony_density.jpg\|thumb\|center\|colony size varies with colony density]]		* As the number of colonies per unit area increases, nutrient diffusion becomes limiting. New molecular biologists are sometimes worried that variation in colony sizes on a plate indicates contamination, but it can be caused by tight packing. [[image:colony_size_varies_with_colony_density.jpg\|thumb\|center\|colony size varies with colony density]]
		+
		+	== Culture Media ==
		+
		+	===Wow.. these 2 batches of TB media are very different in color... did I mess up?===
		+	*A:Maybe, but not necessarily. Different batches of yeast and tryptone can have very different compositions. This is one of the purposes of defined media. [[Image:Two different TB batches.jpg\|thumb\|these two TB batches were made with the same recipe but different bottles of one of the yeast-derived reagents. I forget whether it was yeast extract or tryptone.]]
		+
		+	=== Why do we grow E. coli on glycerol, not glucose? ===
		+	* Amanda Smith found:
		+	**"Glycerol play the part of a cheap carbon-energy source for E.coli.
		+	*** Ecoli grown on glycerol excretes less acetate compared to e coli grown on glucose. (due to changes in the biochemical pathways and slower absorption rate of glycerol). Acetate is bad because it limits the growth of e coli. This is due primarily by methionine synthesis inhibition, which in turn disrupts protein synthesis and alters protein expression (A horror story if you are expressing proteins). Acetate also acidifies the media which also cause other changes in E coli physiology (such as reduced growth rate, altered K/Na ion and amino acid anions concentration. Which also has effects on protein synthesis)"

	==Colony PCR==		==Colony PCR==

Janet B. Matsen: /* What is the ballpark efficiency of electroporation? */

2012-11-18T02:00:23Z

What is the ballpark efficiency of electroporation?

←Older revision		Revision as of 02:00, 18 November 2012
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	=== What is the ballpark efficiency of electroporation? ===		=== What is the ballpark efficiency of electroporation? ===
-	* In the experiment described just above, I calculated that for 0.1 ng of plasmid, the maximum efficiency was 5*10<sup>-6</sup>. I haven't looked up literature values to see how this compares to published statistics. ~~I find this amazing, because it~~ makes Gibson cloning seem even more amazing. Even if you get only a few colonies, there were likely many orders of magnitudes more viable plasmids in the transformation product than that!	+	* In the experiment described just above, I calculated that for 0.1 ng of plasmid, the maximum efficiency was 5*10<sup>-6</sup>. I haven't looked up literature values to see how this compares to published statistics. This statistic makes Gibson cloning seem even more amazing. Even if you get only a few colonies, there were likely many orders of magnitudes more viable plasmids in the transformation product than that!

	== Gene Expression & Plasmids ==		== Gene Expression & Plasmids ==

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

2012-11-18T01:50:28Z

What causes an overloaded colony PCR reaction to fail?

←Older revision		Revision as of 01:50, 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? ===
	*A: Thermostable polymerases require magnesium ([http://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/ link]). It has been suggested that because DNA chealates magnesium, overloading a colony PCR may inhibit the polymerase. (11/2012)		*A: Thermostable polymerases require magnesium ([http://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/ link]). It has been suggested that because DNA chealates magnesium, overloading a colony PCR may inhibit the polymerase. (11/2012)
		+	** I'd love to test that by titrating in extra Mg in a series of overloaded colony PCRs. It is also possible there is a crowding effect or unfavorable interactions with the cell lysate and the polymerase.

	==Primers/Oligos==		==Primers/Oligos==

Janet B. Matsen: /* Colony PCR */

2012-11-18T01:48:26Z

Colony PCR

←Older revision		Revision as of 01:48, 18 November 2012
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	===The gel is dark where I loaded it -- what does this mean? ===		===The gel is dark where I loaded it -- what does this mean? ===
	*A: It probably means you overloaded the PCR with too many cells.		*A: It probably means you overloaded the PCR with too many cells.
		+	===What causes an overloaded colony PCR reaction to fail? ===
		+	*A: Thermostable polymerases require magnesium ([http://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/ link]). It has been suggested that because DNA chealates magnesium, overloading a colony PCR may inhibit the polymerase. (11/2012)

	==Primers/Oligos==		==Primers/Oligos==

Janet B. Matsen: /* Buffers */

2012-11-18T01:38:39Z

Buffers

←Older revision		Revision as of 01:38, 18 November 2012
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	===How do I decide the spacing between a promoter and ribosome binding site? A ribosome binding site & the start codon?===		===How do I decide the spacing between a promoter and ribosome binding site? A ribosome binding site & the start codon?===
	*Recall that a promoter starts making a transcript at the +1 site, no matter the sequence, and that the promoter is only for transcription and the RBS is only for translation. The -10 and -35 locations in the promoter that are significant for binding are numbered relative to this +1 site. You can have any arbitrary sequence before the RBS, and people often include operators for genetic regulation. Or, you can put the ribosome binding site next. This is more standard. The ribosome binding site needs to be correctly spaced from the start codon, and you have to look at the individual RBS to determine it. For some, a BioBrick scar is the perfect distance between the end of the RBS and the start codon; this is useful for making an expression vector like [http://2010.igem.org/Team:Washington/Tools_Created/New_Vectors UW's 2010 iGEM vectors].		*Recall that a promoter starts making a transcript at the +1 site, no matter the sequence, and that the promoter is only for transcription and the RBS is only for translation. The -10 and -35 locations in the promoter that are significant for binding are numbered relative to this +1 site. You can have any arbitrary sequence before the RBS, and people often include operators for genetic regulation. Or, you can put the ribosome binding site next. This is more standard. The ribosome binding site needs to be correctly spaced from the start codon, and you have to look at the individual RBS to determine it. For some, a BioBrick scar is the perfect distance between the end of the RBS and the start codon; this is useful for making an expression vector like [http://2010.igem.org/Team:Washington/Tools_Created/New_Vectors UW's 2010 iGEM vectors].
		+
		+	== Miscellaneous ==
		+	===Is it "media" or "medium"?===
		+	I have recently switched to using "medium" when describing a single type of solid or liquid growth substance.

Janet B. Matsen: /* Why do we disinfect with 70% EtOH? Isn't 100% more potent? */

2012-11-18T01:37:06Z

Why do we disinfect with 70% EtOH? Isn't 100% more potent?

←Older revision		Revision as of 01:37, 18 November 2012
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	==Lab Basics:==		==Lab Basics:==
	===Why do we disinfect with 70% EtOH? Isn't 100% more potent?===		===Why do we disinfect with 70% EtOH? Isn't 100% more potent?===
-	*A: I have read that you don't want to denature the proteins that allow substances like water and ethanol into the cell. If you do, the EtOH can't get into the cytosol. By using 70%, you denature some of the transporter proteins but leave enough to allow more to enter the cell. Also, 70% is cheaper than 100%!	+	*A: I have read that you don't want to denature the proteins that allow substances like water and ethanol into the cell. If you do, the EtOH can't get into the cytosol. By using 70%, you denature some of the transporter proteins but leave enough to allow more to enter the cell. Also, 70% is cheaper than 100%!
		+	* I still use 100% for everything.
		+	* 70% is still concentrated enough to be very flamable!
		+
	===Wow.. these 2 batches of TB media are very different in color... did I mess up?===		===Wow.. these 2 batches of TB media are very different in color... did I mess up?===
	*A:Maybe, but not necessarily. Different batches of yeast and tryptone can have very different compositions. This is one of the purposes of defined media. [[Image:Two different TB batches.jpg\|thumb\|these two TB batches were made with the same recipe but different bottles of one of the yeast-derived reagents. I forget whether it was yeast extract or tryptone.]]		*A:Maybe, but not necessarily. Different batches of yeast and tryptone can have very different compositions. This is one of the purposes of defined media. [[Image:Two different TB batches.jpg\|thumb\|these two TB batches were made with the same recipe but different bottles of one of the yeast-derived reagents. I forget whether it was yeast extract or tryptone.]]

Janet B. Matsen: /* How stable is HEPES? */

2012-11-18T01:20:11Z

How stable is HEPES?

←Older revision		Revision as of 01:20, 18 November 2012
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	* Phosphate buffered saline has MUCH lower concentration of phospate buffer. It is mostly salt.		* Phosphate buffered saline has MUCH lower concentration of phospate buffer. It is mostly salt.
	=== How stable is HEPES? ===		=== How stable is HEPES? ===
-	* Not stable. Make it the day you use it.	+	* Not stable. Make it the day you use it.
-		+

	===How do I decide the spacing between a promoter and ribosome binding site? A ribosome binding site & the start codon?===		===How do I decide the spacing between a promoter and ribosome binding site? A ribosome binding site & the start codon?===
	*Recall that a promoter starts making a transcript at the +1 site, no matter the sequence, and that the promoter is only for transcription and the RBS is only for translation. The -10 and -35 locations in the promoter that are significant for binding are numbered relative to this +1 site. You can have any arbitrary sequence before the RBS, and people often include operators for genetic regulation. Or, you can put the ribosome binding site next. This is more standard. The ribosome binding site needs to be correctly spaced from the start codon, and you have to look at the individual RBS to determine it. For some, a BioBrick scar is the perfect distance between the end of the RBS and the start codon; this is useful for making an expression vector like [http://2010.igem.org/Team:Washington/Tools_Created/New_Vectors UW's 2010 iGEM vectors].		*Recall that a promoter starts making a transcript at the +1 site, no matter the sequence, and that the promoter is only for transcription and the RBS is only for translation. The -10 and -35 locations in the promoter that are significant for binding are numbered relative to this +1 site. You can have any arbitrary sequence before the RBS, and people often include operators for genetic regulation. Or, you can put the ribosome binding site next. This is more standard. The ribosome binding site needs to be correctly spaced from the start codon, and you have to look at the individual RBS to determine it. For some, a BioBrick scar is the perfect distance between the end of the RBS and the start codon; this is useful for making an expression vector like [http://2010.igem.org/Team:Washington/Tools_Created/New_Vectors UW's 2010 iGEM vectors].

Janet B. Matsen: /* How does the density of electrocompetent cells affect transformation efficiency? */

2012-11-18T01:13:56Z

How does the density of electrocompetent cells affect transformation efficiency?

←Older revision		Revision as of 01:13, 18 November 2012
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	=== How does the density of electrocompetent cells affect transformation efficiency?===		=== How does the density of electrocompetent cells affect transformation efficiency?===
-	[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|electroporation: number of colonies versus competent cell density]]	+	[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
		+	* It matters a LOT! The undiluted samples (dilution factor = 0) were harvested ad mid-exponential phase and resuspended in 1/500<sup>ths</sup> of the culture volume. Details of the experiment are [https://docs.google.com/document/d/1btKWgYd1GemMa2mBmg-U72bnu2knSyjf_yGqXo8P1nY/edit here].

	=== What is the ballpark efficiency of electroporation? ===		=== What is the ballpark efficiency of electroporation? ===