Janet B. Matsen:Lab Tips & Tricks - Revision history

Janet B. Matsen: /* Transformations */

2013-03-31T15:18:19Z

Transformations

←Older revision		Revision as of 15:18, 31 March 2013
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	[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]		[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]
	* I plate gibson assemblies (usually low efficiency) with beads.		* I plate gibson assemblies (usually low efficiency) with beads.
-
	=== Outgrowth over the weekend is not recommended ===		=== Outgrowth over the weekend is not recommended ===
	*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!		*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!
	[[Image:2012 02 06-transforming-top10-RoomTemp-LB-Amp-Sm.JPG\|thumb\|center\|contaminants from an over-the-weekend outgrowth of a transformation. Plates have Amp & Sm]]		[[Image:2012 02 06-transforming-top10-RoomTemp-LB-Amp-Sm.JPG\|thumb\|center\|contaminants from an over-the-weekend outgrowth of a transformation. Plates have Amp & Sm]]
		+	=== Make your electrocompetent cells super viscous ===
		+	* Really concentrated cells perform better; I aim to concentrate my culture volume 500x when resuspsending in 10% glycerol.
		+	[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
		+	*My cells clump into a "booger" during recovery (pre-plating) that is mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.

	== Keeping primers, plasmids, strain stocks, & sequencing organized ==		== Keeping primers, plasmids, strain stocks, & sequencing organized ==

Janet B. Matsen: /* Nonunifrom plating is good for minipreps you expect to work decently well */

2013-03-31T15:07:25Z

Nonunifrom plating is good for minipreps you expect to work decently well

←Older revision		Revision as of 15:07, 31 March 2013
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	## Spread cells		## Spread cells
	=== Nonunifrom plating is good for minipreps you expect to work decently well ===		=== Nonunifrom plating is good for minipreps you expect to work decently well ===
-	* I like nonuniform spreading because it will allow you to grab single colonies even if your transformation efficiency is really high or really low.	+	* I like nonuniform spreading because it will allow you to grab single colonies even if your transformation efficiency is really high or really low.
	** If the efficiency is high and you plate it nice and evenly, you can have a lawn across the whole plate making it hard to pick single colonies. If the efficiency is low, you don't need to distribute it evenly anyway because a handful of colonies in a relatively small area of a plate will still give you single colonies you can pick.		** If the efficiency is high and you plate it nice and evenly, you can have a lawn across the whole plate making it hard to pick single colonies. If the efficiency is low, you don't need to distribute it evenly anyway because a handful of colonies in a relatively small area of a plate will still give you single colonies you can pick.
	[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]		[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]
-	* I plate gibson assemblies (usually low efficiency) with beads.	+	* I plate gibson assemblies (usually low efficiency) with beads.
		+
	=== Outgrowth over the weekend is not recommended ===		=== Outgrowth over the weekend is not recommended ===
	*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!		*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!

Janet B. Matsen: /* Transformations */

2013-03-31T15:07:08Z

Transformations

←Older revision		Revision as of 15:07, 31 March 2013
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	==Transformations==		==Transformations==
-	*~~Plate transformations by spreading with~~ a ~~bent 20 uL pipette tip~~, ~~not an ethanol~~/flame sterilized stick, ~~glass beads, etc~~. ~~You have to~~ use ~~the~~ pipette tip ~~anyway to slurp up some of~~ the cells, ~~so just~~ bend it ~~using~~ the ~~inside~~ of the ~~eppendorf tube and use it to spread the~~ cells.	+	=== Plating transformations you expect will work ===
-	** Once I accidentally light a small fire using a flame-sterilized stick, and roller beads are a hassle and hard to ~~get non-uniform spreading of cells with.~~ I like nonuniform spreading because it will allow you to grab single colonies even if your transformation efficiency is really high or really low.	+	* If plating a transformation of miniprepped DNA, you can do two things to save time/mess:
		+	# Plate with wooden spreading sticks
		+	## If you expect decent efficiency, there is no need to use roller beads, a flame sterilized stick, or any other extra tool.
		+	# Just use a pipette tip
		+	## Move the cells to the plate with a pipette tip (works for ~20 uL, but may not be as great for 200 uL)
		+	## bend the pipette tip by pressing it carefully against the sterile side of the plate lid
		+	## Spread cells
		+	=== Nonunifrom plating is good for minipreps you expect to work decently well ===
		+	* I like nonuniform spreading because it will allow you to grab single colonies even if your transformation efficiency is really high or really low.
		+	** If the efficiency is high and you plate it nice and evenly, you can have a lawn across the whole plate making it hard to pick single colonies. If the efficiency is low, you don't need to distribute it evenly anyway because a handful of colonies in a relatively small area of a plate will still give you single colonies you can pick.
	[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]		[[image:nonuniform_plating_of_transformed_cells_is_good.jpg\|thumb\|center\|nonuniform plating of transformed cells is good]]
		+	* I plate gibson assemblies (usually low efficiency) with beads.
		+	=== Outgrowth over the weekend is not recommended ===
	*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!		*Don't [[User:Janet B. Matsen: transformations at room temperature \| transform plates at room temp]] over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination!
	[[Image:2012 02 06-transforming-top10-RoomTemp-LB-Amp-Sm.JPG\|thumb\|center\|contaminants from an over-the-weekend outgrowth of a transformation. Plates have Amp & Sm]]		[[Image:2012 02 06-transforming-top10-RoomTemp-LB-Amp-Sm.JPG\|thumb\|center\|contaminants from an over-the-weekend outgrowth of a transformation. Plates have Amp & Sm]]

Janet B. Matsen: /* Gels */

2013-03-28T19:51:04Z

Gels

←Older revision		Revision as of 19:51, 28 March 2013
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	*Running large volumes on a gel ("overloading" the gel) can cause the band size to run too fast or too slow. If you are running a large amount for purification, include a lane next to the ladder that is not overloaded so you can be confident you know the band size. The following gel ran alright, but a slight shift is visible.		*Running large volumes on a gel ("overloading" the gel) can cause the band size to run too fast or too slow. If you are running a large amount for purification, include a lane next to the ladder that is not overloaded so you can be confident you know the band size. The following gel ran alright, but a slight shift is visible.
	[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]		[[image:janet purification gel.jpg\|thumb\|center\|include small not-overloaded lanes when running purification gels]]
		+	* If you have a voltage source that will stop after a set amount of time, feel free to run your gel as you leave and image it in the morning. Diffusion of bands shouldn't be a big problem unless they are << 500 bp.
		+	** Don't do this if you are going to cut & gel purify the band.
		+	[[image:gel sat 16 hours between photos.jpg\|thumb\|center\|gel sat 16 hours between photos]]

	===Purifying DNA ===		===Purifying DNA ===

Janet B. Matsen: /* Tech Support */

2013-03-28T18:23:22Z

Tech Support

←Older revision		Revision as of 18:23, 28 March 2013
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	** I also like the idea that I will never lose track of a paper I want to tell someone about. It isn't uncommon for someone to tell me about a paper but be unable to find it and send it to me. This should never happen for a dedicated Mendeley user.		** I also like the idea that I will never lose track of a paper I want to tell someone about. It isn't uncommon for someone to tell me about a paper but be unable to find it and send it to me. This should never happen for a dedicated Mendeley user.
	*I'm not thrilled with the word plug-in for inserting references.		*I'm not thrilled with the word plug-in for inserting references.
		+
		+	== Shipping Samples ==
		+	=== Shipping samples containing proteins on dry ice ===
		+	* Beware that shipping samples on dry ice (solid CO2) can affect the pH of the sample. It probably doesn't matter for RNA seq samples, but I know some of us send samples containing protein on dry ice from time to time.
		+	** evidence: http://www.nature.com/nmeth/journal/v10/n4/pdf/nmeth.2409.pdf
		+	*** "Proteins generally exhibit low solubility near their isoelectric point (pI). Therefore, acidic proteins have an increased tendency to aggregate or precipitate as pH falls below physiological levels."
		+	** Interestingly, a pH change did not occur when they stored the samples in a -70oC freezer for 72 hours. The CO2 had time to dissipate. Note the tubes are CO2 permeable!

	== Tech Support ==		== Tech Support ==

Janet B. Matsen: /* Keep an extra clean set up pipette tips */

2013-03-27T19:06:49Z

Keep an extra clean set up pipette tips

←Older revision		Revision as of 19:06, 27 March 2013
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	===Keep an extra clean set up pipette tips ===		===Keep an extra clean set up pipette tips ===
	*I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.		*I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.
		+	** I like that it is inside my bench so nobody can accidentally open it under non-sterile conditions.
	[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]		[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]
	*If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.		*If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.
	** '''Alert:''' Autoclaving (on the dry cycle) loosened the filters so they are not sufficiently held in place. Turning a tip upside down is sufficient to release a tip after autoclaving.		** '''Alert:''' Autoclaving (on the dry cycle) loosened the filters so they are not sufficiently held in place. Turning a tip upside down is sufficient to release a tip after autoclaving.
	** Additionally, Andrew said they always autoclave filters used on the bioreactor in the '''wet''' cycle to prevent damage to the filter.		** Additionally, Andrew said they always autoclave filters used on the bioreactor in the '''wet''' cycle to prevent damage to the filter.
		+	*** I haven't tried to autoclave on the wet cycle yet (3/27/2013), but that may be more useful.
	[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave, but the filters get loosened from the tips.]]		[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave, but the filters get loosened from the tips.]]
	**They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)		**They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)

Janet B. Matsen: /* Keep an extra clean set up pipette tips */

2013-03-27T19:05:09Z

Keep an extra clean set up pipette tips

←Older revision		Revision as of 19:05, 27 March 2013
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	[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]		[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]
	*If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.		*If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.
-	[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave]]	+	** '''Alert:''' Autoclaving (on the dry cycle) loosened the filters so they are not sufficiently held in place. Turning a tip upside down is sufficient to release a tip after autoclaving.
		+	** Additionally, Andrew said they always autoclave filters used on the bioreactor in the '''wet''' cycle to prevent damage to the filter.
		+	[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave, but the filters get loosened from the tips.]]
	**They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)		**They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)

Janet B. Matsen: /* Keep an extra clean set up pipette tips */

2013-03-22T17:33:43Z

Keep an extra clean set up pipette tips

←Older revision		Revision as of 17:33, 22 March 2013
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	===Keep an extra clean set up pipette tips ===		===Keep an extra clean set up pipette tips ===
-	* I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.	+	*I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.
	[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]		[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]
-	* If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.	+	*If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.
	[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave]]		[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave]]
-	** They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)	+	**They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)

	=== Contamination on plates ===		=== Contamination on plates ===

Janet B. Matsen: /* Keep an extra clean set up pipette tips */

2013-03-22T17:27:46Z

Keep an extra clean set up pipette tips

←Older revision		Revision as of 17:27, 22 March 2013
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	* I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.		* I find it useful to keep a super fresh box in the drawer that I only pull out for occasions when sterility is critical. When the less sterile tips on my bench are used up, I remove the sterile sticker from a drawer box and move it to the countertop.
	[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]		[[image:extra sterile pipette tips.jpg\|thumb\|center\|extra sterile pipette tips I keep in my drawer.]]
		+	* If the not-so-sterile tip boxes accumulate, it is ok to autoclave our brand.
		+	[[image:autoclaved Ranin brand Green-Pak Tip-rack Refill can be autoclaved.jpg\|thumb\|center\|Ranin brand "Green-Pak Tip-rack Refill" tips can be autoclaved in the dry cycle in our autoclave]]
		+	** They DO melt in the waste autoclave cycle (Casey the dishwasher said so 3/2013)

	=== Contamination on plates ===		=== Contamination on plates ===

Janet B. Matsen: /* Tech Support Angels & Demons */

2013-02-12T15:46:52Z

Tech Support Angels & Demons

←Older revision		Revision as of 15:46, 12 February 2013
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	*I'm not thrilled with the word plug-in for inserting references.		*I'm not thrilled with the word plug-in for inserting references.

-	== Tech Support ~~Angels & Demons~~ ==	+	== Tech Support ==
	I have a lot of questions, always. The longer I work with a piece of equipment or technique, the more I get into the nitty gritty. Thus it is important for me to have good resources to answer the questions I come up with.		I have a lot of questions, always. The longer I work with a piece of equipment or technique, the more I get into the nitty gritty. Thus it is important for me to have good resources to answer the questions I come up with.
	*It is incredible that in this day and age, manuals aren't available online for some manufacturers.		*It is incredible that in this day and age, manuals aren't available online for some manufacturers.