Koch Lab:Protocols/Microsphere-DNA tethering - Revision history

Steven J. Koch at 06:36, 31 October 2007

2007-10-31T06:36:52Z

←Older revision		Revision as of 06:36, 31 October 2007
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	*anti-fluorescein --- fluoresceinDNA		*anti-fluorescein --- fluoresceinDNA

-	~~==Some general thoughts--not rigorously tested==~~
	==Specific protocols==		==Specific protocols==
-	*~~A specific protocol~~, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres	+	*[[/Glass, dig, biotin, microsphere, 4kb DNA\|4411 basepair PCR-labeled DNA (dig, biotin), 0.5 micron streptavidin microspheres]]

	</div>		</div>
	[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]		[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]
	[[Category:Koch Lab Page]]		[[Category:Koch Lab Page]]

Steven J. Koch: /* Principle */

2007-10-31T06:34:41Z

Principle

←Older revision		Revision as of 06:34, 31 October 2007
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	<div style="padding: 10px; width: 720px; border: 5px solid #008;">		<div style="padding: 10px; width: 720px; border: 5px solid #008;">
	==Principle==		==Principle==
-	[[Image:Glass-DNA-Microsphere Tether.png\|right\|thumb\|]]	+	[[Image:Glass-DNA-Microsphere Tether.png\|right\|thumb\|Schematic of microsphere-DNA tethering]]
	A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).		A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).

Steven J. Koch: /* Principle */

2007-10-31T06:34:14Z

Principle

←Older revision		Revision as of 06:34, 31 October 2007
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	<div style="padding: 10px; width: 720px; border: 5px solid #008;">		<div style="padding: 10px; width: 720px; border: 5px solid #008;">
	==Principle==		==Principle==
		+	[[Image:Glass-DNA-Microsphere Tether.png\|right\|thumb\|]]
	A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).		A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).

Steven J. Koch: /* Principle */

2007-10-31T06:29:54Z

Principle

←Older revision		Revision as of 06:29, 31 October 2007
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	glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere		glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere

-	There are also many other methods. Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular. Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are: anti-HA --- HA-RNA polymerase-DNA; gold --- thiol-DNA; anti-his --- his-Protein-DNA; anti-fluorescein --- fluoresceinDNA	+	There are also many other methods. Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular. Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are:
		+	*anti-HA --- HA-RNA polymerase-DNA
		+	*gold --- thiol-DNA
		+	*anti-his --- his-Protein-DNA
		+	*anti-fluorescein --- fluoresceinDNA

	==Some general thoughts--not rigorously tested==		==Some general thoughts--not rigorously tested==

Steven J. Koch: /* Principle */

2007-10-31T06:29:18Z

Principle

←Older revision		Revision as of 06:29, 31 October 2007
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	<div style="padding: 10px; width: 720px; border: 5px solid #008;">		<div style="padding: 10px; width: 720px; border: 5px solid #008;">
	==Principle==		==Principle==
		+	A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).
		+
		+	To achieve tethering, you need to attach one end of the DNA to the glass slide, and the other end to the microsphere. Generically, this is what needs to be done:
		+	glass --- connection#1 --- endDNA******DNAend --- connection#2 --- microsphere
		+
		+	A very common method that is used is to label the DNA with [http://en.wikipedia.org/wiki/Digoxigenin digoxigenin (dig)] on one end and [http://en.wikipedia.org/wiki/Biotin biotin] on the other end. The surface of the glass slide is coated with a polyclonal antibody against dig (anti-dig) and the microspheres are coated with streptavidin (which binds to biotin with high affinity). This makes the tethering scheme:
		+	glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere
		+
		+	There are also many other methods. Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular. Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are: anti-HA --- HA-RNA polymerase-DNA; gold --- thiol-DNA; anti-his --- his-Protein-DNA; anti-fluorescein --- fluoresceinDNA
		+
	==Some general thoughts--not rigorously tested==		==Some general thoughts--not rigorously tested==
	==Specific protocols==		==Specific protocols==

Steven J. Koch at 05:59, 31 October 2007

2007-10-31T05:59:58Z

←Older revision		Revision as of 05:59, 31 October 2007
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	==Principle==		==Principle==
	==Some general thoughts--not rigorously tested==		==Some general thoughts--not rigorously tested==
-	==A specific protocol, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres==	+	==Specific protocols==
		+	*A specific protocol, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres

	</div>		</div>
	[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]		[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]
	[[Category:Koch Lab Page]]		[[Category:Koch Lab Page]]

Steven J. Koch at 05:59, 31 October 2007

2007-10-31T05:59:24Z

←Older revision		Revision as of 05:59, 31 October 2007
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	</div>		</div>
-	[[Category:Protocol]] [[Category:DNA]]	+	[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]
	[[Category:Koch Lab Page]]		[[Category:Koch Lab Page]]

Steven J. Koch: New page: {{Koch_Lab}}
==Principle== ==Some general thoughts--not rigorously tested== ==A specific protocol, 4411 basepair PCR-labe...

2007-10-31T05:57:43Z

New page: {{Koch_Lab}} <div style="padding: 10px; width: 720px; border: 5px solid #008;"> ==Principle== ==Some general thoughts--not rigorously tested== ==A specific protocol, 4411 basepair PCR-labe...

New page

{{Koch_Lab}}
<div style="padding: 10px; width: 720px; border: 5px solid #008;">
==Principle==
==Some general thoughts--not rigorously tested==
==A specific protocol, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres==

</div>
[[Category:Protocol]] [[Category:DNA]]
[[Category:Koch Lab Page]]

Koch Lab:Protocols/Microsphere-DNA tethering - Revision history

Steven J. Koch at 06:36, 31 October 2007

Steven J. Koch: /* Principle */

Steven J. Koch: /* Principle */

Steven J. Koch: /* Principle */

Steven J. Koch: /* Principle */

Steven J. Koch at 05:59, 31 October 2007

Steven J. Koch at 05:59, 31 October 2007

Steven J. Koch: New page: {{Koch_Lab}} ==Principle== ==Some general thoughts--not rigorously tested== ==A specific protocol, 4411 basepair PCR-labe...

Steven J. Koch: New page: {{Koch_Lab}}
==Principle== ==Some general thoughts--not rigorously tested== ==A specific protocol, 4411 basepair PCR-labe...