Library Generation - Revision history

Torsten Waldminghaus at 17:34, 23 February 2009

2009-02-23T17:34:55Z

←Older revision		Revision as of 17:34, 23 February 2009
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		+	{{back to protocols}}
	[[User:Smoore\|Smoore]]		[[User:Smoore\|Smoore]]
	==Background==		==Background==

Jenny T Nguyen at 18:50, 10 July 2006

2006-07-10T18:50:10Z

←Older revision		Revision as of 18:50, 10 July 2006
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	Be aware that there are a lot of ways to overcome selective pressure that you may not have thought of. If the cell finds a way to alter the copy number of a plasmid, or up-regulate a toxin exporter, they will look like good clones, but have nothing to do with what you were looking for.		Be aware that there are a lot of ways to overcome selective pressure that you may not have thought of. If the cell finds a way to alter the copy number of a plasmid, or up-regulate a toxin exporter, they will look like good clones, but have nothing to do with what you were looking for.
		+
		+	[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Smoore at 20:01, 6 October 2005

2005-10-06T20:01:52Z

←Older revision		Revision as of 20:01, 6 October 2005
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		+	[[User:Smoore\|Smoore]]
	==Background==		==Background==
	Genetic libraries are collections of genes present in some recombinant DNA form so they can be propagated. When people refer to “screening a library” they usually have some phenotype that they are able to select or screen for and evaluate a large number of library clones to look for a gene that alters the phenotype. People interested in eukaryotic biology usually make cDNA libraries that are derived from pools of mRNA isolated from an organism of interest. This allows them to isolate DNA fragments that encode proteins or RNA that are produced form the spliced form of the RNAs found in the cells. It has been a long time since I have worked with cDNA libraries, so I won’t go into that here (perhaps someone in another group can add a section?).		Genetic libraries are collections of genes present in some recombinant DNA form so they can be propagated. When people refer to “screening a library” they usually have some phenotype that they are able to select or screen for and evaluate a large number of library clones to look for a gene that alters the phenotype. People interested in eukaryotic biology usually make cDNA libraries that are derived from pools of mRNA isolated from an organism of interest. This allows them to isolate DNA fragments that encode proteins or RNA that are produced form the spliced form of the RNAs found in the cells. It has been a long time since I have worked with cDNA libraries, so I won’t go into that here (perhaps someone in another group can add a section?).

Smoore: /* Genomic DNA Preparation */

2005-10-06T19:58:28Z

Genomic DNA Preparation

←Older revision		Revision as of 19:58, 6 October 2005
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	*Dry the pellets by setting the tubes on their side.		*Dry the pellets by setting the tubes on their side.
	*Resuspend and pool the DNA in <b>100 μ TE</b>. This is 200 X the original culture volume. Assuming each gene was present in one copy per cell (an under-estimate), you’ll have about 6 X 10<sup>10</sup> copies of each gene in your tube. Plenty.		*Resuspend and pool the DNA in <b>100 μ TE</b>. This is 200 X the original culture volume. Assuming each gene was present in one copy per cell (an under-estimate), you’ll have about 6 X 10<sup>10</sup> copies of each gene in your tube. Plenty.
-	*Determine the concentration of DNA using absorbance at 260 nm.	+	*Determine the concentration of DNA using absorbance at 260 nm. The DNA prep will most likely still have RNA fragemts in it, don't sweat it, but keep in mind that they also add to the absorbance reading.

	===Digestion and Ligation===		===Digestion and Ligation===

Smoore: /* Recipes */

2005-10-06T19:56:35Z

Recipes

←Older revision		Revision as of 19:56, 6 October 2005
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-	<b>TE Buffer</b>(Tris-EDTA)	+	<b>TE Buffer</b> (Tris-EDTA)

	10 mM Tris-Cl, pH 8.0		10 mM Tris-Cl, pH 8.0

Smoore: /* Recipes */

2005-10-06T19:56:13Z

Recipes

←Older revision		Revision as of 19:56, 6 October 2005
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	1 μg/mL RNase A		1 μg/mL RNase A
		+

	<b>TE Buffer</b>(Tris-EDTA)		<b>TE Buffer</b>(Tris-EDTA)

Smoore: /* Recipes */

2005-10-06T19:55:44Z

Recipes

←Older revision		Revision as of 19:55, 6 October 2005
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	1 mM EDTA		1 mM EDTA
		+

	*You can use other detergents instead of B-Per, that's what I use. Bug-Buster is another commercial detergent that doesn't disrupt proteins. There are protocols for using Tween, NP-40, and Triton as well. You just need to get the outer membranr disrupted so the lysozyme has access. I have had good luck with ~1-2% chloroform as well.		*You can use other detergents instead of B-Per, that's what I use. Bug-Buster is another commercial detergent that doesn't disrupt proteins. There are protocols for using Tween, NP-40, and Triton as well. You just need to get the outer membranr disrupted so the lysozyme has access. I have had good luck with ~1-2% chloroform as well.
		+

	<b>10% CTAB solution</b>		<b>10% CTAB solution</b>
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	700 mM NaCl		700 mM NaCl
-
-
-	~~<b></b>~~
-	~~<b></b>~~

	===Transformation and Plating===		===Transformation and Plating===

Smoore: /* Protocol */

2005-10-06T19:54:57Z

Protocol

←Older revision		Revision as of 19:54, 6 October 2005
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	1 mM EDTA		1 mM EDTA
		+
		+	*You can use other detergents instead of B-Per, that's what I use. Bug-Buster is another commercial detergent that doesn't disrupt proteins. There are protocols for using Tween, NP-40, and Triton as well. You just need to get the outer membranr disrupted so the lysozyme has access. I have had good luck with ~1-2% chloroform as well.
		+
		+	<b>10% CTAB solution</b>
		+
		+	10% CTAB (some crazy name that doesn't start with a "C")
		+
		+	700 mM NaCl


-	~~<b></b>~~
	<b></b>		<b></b>
	<b></b>		<b></b>

Smoore: /* Recipes */

2005-10-06T19:49:08Z

Recipes

←Older revision		Revision as of 19:49, 6 October 2005
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	*Transform each test ligation, you can probably plate the whole thing on one plate. Count the colonies that form and use the insert-to-vector ratio that gave the most colonies to set up the large ligation reactions.		*Transform each test ligation, you can probably plate the whole thing on one plate. Count the colonies that form and use the insert-to-vector ratio that gave the most colonies to set up the large ligation reactions.

-	====Recipes====	+	====<u>Recipes</u>====
		+
	<b>Rinse Buffer</b>		<b>Rinse Buffer</b>
		+
	10 mM Tris-Cl, pH 8.0		10 mM Tris-Cl, pH 8.0
		+
	100 mM NaCl		100 mM NaCl
		+

	<b>Lysis Buffer</b>		<b>Lysis Buffer</b>
		+
	B-Per (Pierce) supplemented with:		B-Per (Pierce) supplemented with:
		+
	5 mM EDTA (from neutral pH stock)		5 mM EDTA (from neutral pH stock)
		+
	0.1 mg/mL Lysozyme (from fresh 10 mg/mL stock in PBS or TBS)		0.1 mg/mL Lysozyme (from fresh 10 mg/mL stock in PBS or TBS)
		+
		+	1 μg/mL RNase A

		+	<b>TE Buffer</b>(Tris-EDTA)
		+
		+	10 mM Tris-Cl, pH 8.0
		+
		+	1 mM EDTA
		+

	<b></b>		<b></b>
	<b></b>		<b></b>
	<b></b>		<b></b>
-	~~<b></b>~~
-

	===Transformation and Plating===		===Transformation and Plating===

Smoore at 19:38, 6 October 2005

2005-10-06T19:38:42Z

←Older revision		Revision as of 19:38, 6 October 2005
Line 67:		Line 67:

	*Transform each test ligation, you can probably plate the whole thing on one plate. Count the colonies that form and use the insert-to-vector ratio that gave the most colonies to set up the large ligation reactions.		*Transform each test ligation, you can probably plate the whole thing on one plate. Count the colonies that form and use the insert-to-vector ratio that gave the most colonies to set up the large ligation reactions.
		+
		+	====Recipes====
		+	<b>Rinse Buffer</b>
		+	10 mM Tris-Cl, pH 8.0
		+	100 mM NaCl
		+
		+	<b>Lysis Buffer</b>
		+	B-Per (Pierce) supplemented with:
		+	5 mM EDTA (from neutral pH stock)
		+	0.1 mg/mL Lysozyme (from fresh 10 mg/mL stock in PBS or TBS)
		+
		+
		+	<b></b>
		+	<b></b>
		+	<b></b>
		+	<b></b>
		+

	===Transformation and Plating===		===Transformation and Plating===