User:Pranav Rathi/Notebook/OT/2012/10/01/Buffer preparation for DNA overstretching & unzipping experiments - Revision history

Pranav Rathi: /* Procedure */

2013-01-04T23:16:08Z

Procedure

←Older revision		Revision as of 23:16, 4 January 2013
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	# First weight out 15mg of BGB poweder		# First weight out 15mg of BGB poweder
-	# Add ~~1.5~~ ml of 1x pop and mix by vortexing	+	# Add 3 ml of 1x pop and mix by vortexing
	# Run through .2μM syringe filter using 3ml syringe.		# Run through .2μM syringe filter using 3ml syringe.
	# Now store 5mg/ml BGB in 1x pop buffer (minus amount of protein that stuck to filter) at 3C.		# Now store 5mg/ml BGB in 1x pop buffer (minus amount of protein that stuck to filter) at 3C.

2013-01-04T22:50:30Z

Anti-Dig Solution

←Older revision		Revision as of 22:50, 4 January 2013
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	# Make 20μgm/ml of aliquots.		# Make 20μgm/ml of aliquots.
	# Nitrogen flash-freeze them and store them at -80C.		# Nitrogen flash-freeze them and store them at -80C.
-	# When use for sample; take out an aliquot from freezer and mix ~~180mL~~ of cold PBS and store at +3C.	+	# When use for sample; take out an aliquot from freezer and mix 180μL of cold PBS and store at +3C.

	=== Microspheres Solution===		=== Microspheres Solution===

2012-10-16T14:26:47Z

Procedure

←Older revision		Revision as of 14:26, 16 October 2012
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-	This page discussed the general procedure used to make different buffers and solutions. Normally solutions ~~depends~~ on the specific sample, but the buffers;POP and PBS ~~remains~~ similar as discussed.	+	This page discussed the general procedure used to make different buffers and solutions. Normally solutions depend on the specific sample, but the buffers;POP and PBS remain similar as discussed.

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2012-10-16T14:23:44Z

←Older revision		Revision as of 14:23, 16 October 2012
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-	We primarily use popping buffer and PBS for DNA-tethering, unzipping and overstretching experiments. All the other buffers consist these two. This page discusses the chemicals and process used in preparation of the buffers and solutions.	+	We primarily use popping buffer and PBS for DNA-tethering, unzipping and overstretching experiments. All the other buffers consist of these two. This page discusses the chemicals and process used in preparation of the buffers and solutions.

	===Popping Buffer===		===Popping Buffer===

2012-10-16T01:41:19Z

Destroying Clumps

←Older revision		Revision as of 01:41, 16 October 2012
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	----		----

-	In tests done, it seems this method works as well ~~(or as poor depending on your perspective) as sonicating~~. Vortex in pulses of 5 seconds for however long you deem worthy depending on your volume and level of bead clumps.	+	In tests done, it seems this method works as well, but i never tried it. Vortex in pulses of 5 seconds for however long you deem worthy depending on your volume and level of bead clumps.
	<gallery>		<gallery>
	Image:Halfmicron bead sonicated.png\|This image was taken after sonication.		Image:Halfmicron bead sonicated.png\|This image was taken after sonication.

2012-10-16T01:40:13Z

Destroying Clumps

←Older revision		Revision as of 01:40, 16 October 2012
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	</gallery>		</gallery>
-	Based on these images, ~~it seems like not washing~~ is ~~the best possible choice because the~~ beads ~~exist in solution as single entities~~.	+	Based on these images, there is no clumping if beads are sonicated for sufficient time.
	----		----
	# Takeout 50μL from bottle to keep in stock		# Takeout 50μL from bottle to keep in stock

2012-10-16T01:38:41Z

Destroying Clumps

←Older revision		Revision as of 01:38, 16 October 2012
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	In tests done, it seems this method works as well (or as poor depending on your perspective) as sonicating. Vortex in pulses of 5 seconds for however long you deem worthy depending on your volume and level of bead clumps.		In tests done, it seems this method works as well (or as poor depending on your perspective) as sonicating. Vortex in pulses of 5 seconds for however long you deem worthy depending on your volume and level of bead clumps.
	<gallery>		<gallery>
-	Image:~~Sonicate~~.png\|This image was taken after sonication.	+	Image:Halfmicron bead sonicated.png\|This image was taken after sonication.
-	Image:~~Vortex2~~.png\|This image was taken ~~after vortexing (instead of sonicating). Seems as clumpy as sonicating.~~	+	Image:Onemicron bead sonicated.png\|This image was taken after sonication.
-	~~Image:Top vortex.png\|This is a view of one surface of the sample in vortexing, but the same can be seen~~ after sonication.	+
-	~~Image:Nowashing.png\|Instead of washing and sonicating/vortexing I just diluted the stock beads in water. Not dilute enough, but no clumps.~~	+
	</gallery>		</gallery>
	Based on these images, it seems like not washing is the best possible choice because the beads exist in solution as single entities.		Based on these images, it seems like not washing is the best possible choice because the beads exist in solution as single entities.

2012-10-16T01:31:59Z

Destroying Clumps

←Older revision		Revision as of 01:31, 16 October 2012
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-	You will want to sonicate beads for a decent amount of time. The time it takes depends on the size of the beads and the level of clumpiness. Also adding buffer that reduces the hydrophobicity of the bead surface may be of use. I usually sonicate the beads for 180 seconds for both sizes.	+	You will want to sonicate beads for a decent amount of time. The time it takes depends on the size of the beads and the level of clumpiness. Also adding buffer that reduces the hydrophobicity of the bead surface may be of use. I usually sonicate the beads for 180 seconds for both sizes. The results are great no clumping as shown below; in picture beads are 1:10 diluted.

2012-10-15T20:23:35Z

Destroying Clumps

←Older revision		Revision as of 20:23, 15 October 2012
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	----		----

-	You will want to sonicate beads for a decent amount of time. The time it takes depends on the size of the beads and the level of clumpiness. Also adding buffer that reduces the hydrophobicity of the bead surface may be of use.	+	You will want to sonicate beads for a decent amount of time. The time it takes depends on the size of the beads and the level of clumpiness. Also adding buffer that reduces the hydrophobicity of the bead surface may be of use. I usually sonicate the beads for 180 seconds for both sizes.

2012-10-15T20:22:20Z

Bangs Labs Method

←Older revision		Revision as of 20:22, 15 October 2012
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	#Discard supernatant.		#Discard supernatant.
	#Repeat steps 2-3 for a total of 3-5 times		#Repeat steps 2-3 for a total of 3-5 times
-	#After discarding of supernatant, ~~add 250ul of BGB+Popping Buffer~~	+	#After discarding of supernatant, Same as above.
		+
	======Destroying Clumps======		======Destroying Clumps======
	[[Image:Before.png\|thumb\|Clumps of beads can easily be seen after washing. It is recommended one destroy these after washing.]] It is important to get beads to be suspended individually because clumpiness can really ruin a sample. Below are two ways that this can be achieved. With proof.		[[Image:Before.png\|thumb\|Clumps of beads can easily be seen after washing. It is recommended one destroy these after washing.]] It is important to get beads to be suspended individually because clumpiness can really ruin a sample. Below are two ways that this can be achieved. With proof.