Purification of DNA - Revision history

Christopher C Vanlang: /* See also */

Christopher C Vanlang — Fri, 30 Dec 2011 01:55:49 GMT

Christopher C Vanlang: /* Alcohol precipitation */

Christopher C Vanlang — Thu, 22 Dec 2011 09:37:31 GMT

Alcohol precipitation

←Older revision		Revision as of 09:37, 22 December 2011
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	==Alcohol precipitation==		==Alcohol precipitation==
		+	#[[DNA Precipitation]]
	#[[Ethanol precipitation of nucleic acids]]		#[[Ethanol precipitation of nucleic acids]]
	#[[Ethanol precipitation of small DNA fragments]]		#[[Ethanol precipitation of small DNA fragments]]

Jakob Suckale: links

Jakob Suckale — Wed, 07 Jul 2010 12:00:39 GMT

links

←Older revision		Revision as of 12:00, 7 July 2010
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	#[[QIAquick PCR purification]]		#[[QIAquick PCR purification]]
	#*for typical DNA fragments (> 200 bp in length)		#*for typical DNA fragments (> 200 bp in length)
-
-
-	~~[[Category:Protocol]]~~
-	~~[[Category:DNA]]~~
-	~~[[Category:In vitro]]~~
-
-

	Products from the restriction need to a number of reagents removed from the previous restriction. These include salts (from buffers) and restriction enzymes. DNA can be removed and washed from solution by using column purification kits. Here is a rough description of how the kit works.		Products from the restriction need to a number of reagents removed from the previous restriction. These include salts (from buffers) and restriction enzymes. DNA can be removed and washed from solution by using column purification kits. Here is a rough description of how the kit works.
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	Step 5: Elution. Elution buffer is Tris buffer with some EDTA at a pH of 8-8.5. EDTA binds to divalent cations, particularly magnesium (Mg2+). Tris has labile protons with a pKa of 8.30 (at 20 °C; this declines approximately 0.03 units per degree Celsius rise in temperature). Tris is often used when working with nucleic acids. Tris is an effective buffer for slightly basic solutions, which keeps DNA deprotonated and soluble in water. These ions are necessary co-factors for many enzymes; Magnesium is a co-factor for many DNA-modifying enzymes. Tris is toxic to mammalian cells, and reacts strongly with pH electrodes. It is a primary amine, and can thus react with aldehydes.		Step 5: Elution. Elution buffer is Tris buffer with some EDTA at a pH of 8-8.5. EDTA binds to divalent cations, particularly magnesium (Mg2+). Tris has labile protons with a pKa of 8.30 (at 20 °C; this declines approximately 0.03 units per degree Celsius rise in temperature). Tris is often used when working with nucleic acids. Tris is an effective buffer for slightly basic solutions, which keeps DNA deprotonated and soluble in water. These ions are necessary co-factors for many enzymes; Magnesium is a co-factor for many DNA-modifying enzymes. Tris is toxic to mammalian cells, and reacts strongly with pH electrodes. It is a primary amine, and can thus react with aldehydes.

-	DNA is now stabilized and ready for long-term storage at -20C.	+	DNA is now stabilized and ready for long-term storage at -20C.
		+
		+	== See also ==
		+	* [[PCR inhibitors]]
		+
		+	[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Kersten S. Rabe: /* Size-dependent precipitation by PEG8000/MgCl2 */

Kersten S. Rabe — Wed, 27 Aug 2008 23:05:41 GMT

Size-dependent precipitation by PEG8000/MgCl2

←Older revision		Revision as of 23:05, 27 August 2008
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	==Size-dependent precipitation by PEG8000/MgCl<sub>2</sub>==		==Size-dependent precipitation by PEG8000/MgCl<sub>2</sub>==
	#[[Protocol Size selective DNA precipitation by PEG/MgCl2]]		#[[Protocol Size selective DNA precipitation by PEG/MgCl2]]
-	*A very easy and fast way to remove e.g. primers for downstream applications	+	A very easy and fast way to remove e.g. primers for downstream applications

	==Filtration / affinity columns==		==Filtration / affinity columns==

Kersten S. Rabe: /* Size-dependent precipitation by PEG8000/MgCl2 */

Kersten S. Rabe — Wed, 27 Aug 2008 23:05:27 GMT

Size-dependent precipitation by PEG8000/MgCl2

←Older revision		Revision as of 23:05, 27 August 2008
Line 14:		Line 14:
	==Size-dependent precipitation by PEG8000/MgCl<sub>2</sub>==		==Size-dependent precipitation by PEG8000/MgCl<sub>2</sub>==
	#[[Protocol Size selective DNA precipitation by PEG/MgCl2]]		#[[Protocol Size selective DNA precipitation by PEG/MgCl2]]
		+	*A very easy and fast way to remove e.g. primers for downstream applications

	==Filtration / affinity columns==		==Filtration / affinity columns==

Kersten S. Rabe at 23:04, 27 August 2008

Kersten S. Rabe — Wed, 27 Aug 2008 23:04:35 GMT

←Older revision		Revision as of 23:04, 27 August 2008
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	2-propanol (isopropanol) precipitation yields less DNA than EtOH precipitation. (In my hands 60% less on average! [[User:Jasu\|Jasu]])		2-propanol (isopropanol) precipitation yields less DNA than EtOH precipitation. (In my hands 60% less on average! [[User:Jasu\|Jasu]])
	2-propanol at RT reduces the risk of co-precipitation of salts (which can interfere with downstream experiments). 2-propanol is preferred for large volumes of DNA since less alcohol is required, plus it's faster since it doesn't require a cooling step. Glassy pellets from 2-propanol precipitation are harder to see than fluffy, salt-containing pellets from ethanol precipitation. 2-propanol pellets are also more loosely attached after centrifugation. Take care when decanting.		2-propanol at RT reduces the risk of co-precipitation of salts (which can interfere with downstream experiments). 2-propanol is preferred for large volumes of DNA since less alcohol is required, plus it's faster since it doesn't require a cooling step. Glassy pellets from 2-propanol precipitation are harder to see than fluffy, salt-containing pellets from ethanol precipitation. 2-propanol pellets are also more loosely attached after centrifugation. Take care when decanting.
		+
		+	==Size-dependent precipitation by PEG8000/MgCl<sub>2</sub>==
		+	#[[Protocol Size selective DNA precipitation by PEG/MgCl2]]

	==Filtration / affinity columns==		==Filtration / affinity columns==

Melissali: /* Filtration / affinity columns */

Melissali — Thu, 20 Sep 2007 15:34:51 GMT

Filtration / affinity columns

←Older revision		Revision as of 15:34, 20 September 2007
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	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]
		+
		+

	Products from the restriction need to a number of reagents removed from the previous restriction. These include salts (from buffers) and restriction enzymes. DNA can be removed and washed from solution by using column purification kits. Here is a rough description of how the kit works.		Products from the restriction need to a number of reagents removed from the previous restriction. These include salts (from buffers) and restriction enzymes. DNA can be removed and washed from solution by using column purification kits. Here is a rough description of how the kit works.

Melissali: /* Filtration / affinity columns */

Melissali — Thu, 20 Sep 2007 15:33:55 GMT

Filtration / affinity columns

←Older revision		Revision as of 15:33, 20 September 2007
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	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]
		+
		+	Products from the restriction need to a number of reagents removed from the previous restriction. These include salts (from buffers) and restriction enzymes. DNA can be removed and washed from solution by using column purification kits. Here is a rough description of how the kit works.
		+
		+	Step 1: Enhance DNA negative charge. DNA is negatively charged because of the phosphate backbone, so this step enhances the negative charge. In the Qiagen kit, the PB solution contains guanine chloride, a protein detergent and denaturant. The solution is also slightly acidic due to the low pH
		+
		+	Step 2: Stick the negative DNA to the positively charged resin. Load your negatively charged DNA product into the tube and centrifuge it (usually around 14,000 rpm for 1 min).
		+
		+	Step 3: Wash DNA to remove small pieces of DNA. This is achieved using Ethanol and Tris buffer and is labelled as the “PE” buffer in the Qiagen kit. This solution solubilizes smaller pieces of DNA so that they can be eluted from the column. Generally you add a large amount of this stuff and spin the column (~750mL of PE buffer, spin for 1 min).
		+
		+	Step 4: Remove excess EtOH. After dumping out the EtOH from the previous step, another spin is good to remove all the EtOH from the column and DNA so that it doesn't get into the final eluted product. Spin for about 12 minutes at 14,000 rpm).
		+
		+	Step 5: Elution. Elution buffer is Tris buffer with some EDTA at a pH of 8-8.5. EDTA binds to divalent cations, particularly magnesium (Mg2+). Tris has labile protons with a pKa of 8.30 (at 20 °C; this declines approximately 0.03 units per degree Celsius rise in temperature). Tris is often used when working with nucleic acids. Tris is an effective buffer for slightly basic solutions, which keeps DNA deprotonated and soluble in water. These ions are necessary co-factors for many enzymes; Magnesium is a co-factor for many DNA-modifying enzymes. Tris is toxic to mammalian cells, and reacts strongly with pH electrodes. It is a primary amine, and can thus react with aldehydes.
		+
		+	DNA is now stabilized and ready for long-term storage at -20C.

Jakob Suckale: /* Alcohol precipitation */ comparison EtOH 2PrOH

Jakob Suckale — Wed, 25 Apr 2007 11:15:52 GMT

Alcohol precipitation: comparison EtOH 2PrOH

←Older revision		Revision as of 11:15, 25 April 2007
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	#[[Isopropanol Precipitation for PCR Purification]]		#[[Isopropanol Precipitation for PCR Purification]]
	#*use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns		#*use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns
		+
		+	2-propanol (isopropanol) precipitation yields less DNA than EtOH precipitation. (In my hands 60% less on average! [[User:Jasu\|Jasu]])
		+	2-propanol at RT reduces the risk of co-precipitation of salts (which can interfere with downstream experiments). 2-propanol is preferred for large volumes of DNA since less alcohol is required, plus it's faster since it doesn't require a cooling step. Glassy pellets from 2-propanol precipitation are harder to see than fluffy, salt-containing pellets from ethanol precipitation. 2-propanol pellets are also more loosely attached after centrifugation. Take care when decanting.

	==Filtration / affinity columns==		==Filtration / affinity columns==

Jakob Suckale: header level increased

Jakob Suckale — Wed, 25 Apr 2007 11:00:01 GMT

header level increased

←Older revision		Revision as of 11:00, 25 April 2007
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	There are several methods for purifying DNA. The one you choose depends on the nature of your DNA sample and your downstream applications.		There are several methods for purifying DNA. The one you choose depends on the nature of your DNA sample and your downstream applications.

-	===Alcohol precipitation===	+	==Alcohol precipitation==
	#[[Ethanol precipitation of nucleic acids]]		#[[Ethanol precipitation of nucleic acids]]
	#[[Ethanol precipitation of small DNA fragments]]		#[[Ethanol precipitation of small DNA fragments]]
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	#*use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns		#*use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns

-	===Filtration / affinity columns===	+	==Filtration / affinity columns==
	#[[Centrifugal filtration/Nucleic acids]]		#[[Centrifugal filtration/Nucleic acids]]
	#*for small DNA fragments (~50-200 bp in length)		#*for small DNA fragments (~50-200 bp in length)

←Older revision		Revision as of 01:55, 30 December 2011
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	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]
		+	[[Category:Purification]]