Resuspension of primers - Revision history

Barry Canton: Reconstituting primers moved to Resuspension of primers

Barry Canton — Tue, 16 Sep 2008 00:06:19 GMT

Reconstituting primers moved to Resuspension of primers

←Older revision

Revision as of 00:06, 16 September 2008

Jenny T Nguyen at 18:54, 10 July 2006

Jenny T Nguyen — Mon, 10 Jul 2006 18:54:43 GMT

←Older revision		Revision as of 18:54, 10 July 2006
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	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the [[EDTA]] in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each [[EDTA]] molecule chelates one Mg<sup>2+</sup> ion.		However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the [[EDTA]] in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each [[EDTA]] molecule chelates one Mg<sup>2+</sup> ion.
	*You typically want a small amount of [[EDTA]] around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by [[EDTA]] than Mg. That said, I typically resuspend all of my oligos in [[TE buffer]] (mine is 10 mM Tris-HCl, pH 7.5, 1 mM [[EDTA]]) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM [[EDTA]] final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about [[EDTA]], including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]		*You typically want a small amount of [[EDTA]] around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by [[EDTA]] than Mg. That said, I typically resuspend all of my oligos in [[TE buffer]] (mine is 10 mM Tris-HCl, pH 7.5, 1 mM [[EDTA]]) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM [[EDTA]] final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about [[EDTA]], including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]
		+
		+	[[Category:Protocol]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Barry Canton: /* Method */

Barry Canton — Tue, 07 Mar 2006 18:31:16 GMT

Method

←Older revision		Revision as of 18:31, 7 March 2006
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	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the [[EDTA]] in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each [[EDTA]] molecule chelates one Mg<sup>2+</sup> ion.		However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the [[EDTA]] in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each [[EDTA]] molecule chelates one Mg<sup>2+</sup> ion.
-	*You typically want a small amount of [[EDTA]] around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by [[EDTA]] than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM [[EDTA]]) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM [[EDTA]] final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about [[EDTA]], including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]	+	*You typically want a small amount of [[EDTA]] around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by [[EDTA]] than Mg. That said, I typically resuspend all of my oligos in [[TE buffer]] (mine is 10 mM Tris-HCl, pH 7.5, 1 mM [[EDTA]]) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM [[EDTA]] final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about [[EDTA]], including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]

Jason R. Kelly at 01:07, 5 January 2006

Jason R. Kelly — Thu, 05 Jan 2006 01:07:14 GMT

←Older revision		Revision as of 01:07, 5 January 2006
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	*Allow to sit for 2mins, then vortex for 15s.		*Allow to sit for 2mins, then vortex for 15s.

-	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.	+	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the [[EDTA]] in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each [[EDTA]] molecule chelates one Mg<sup>2+</sup> ion.
-	*You typically want a small amount of EDTA around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by EDTA than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM EDTA final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about EDTA, including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]	+	*You typically want a small amount of [[EDTA]] around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by [[EDTA]] than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM [[EDTA]]) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM [[EDTA]] final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about [[EDTA]], including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]

Kathmc at 21:52, 4 January 2006

Kathmc — Wed, 04 Jan 2006 21:52:23 GMT

←Older revision		Revision as of 21:52, 4 January 2006
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	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.		However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.
-	*You typically want a small amount of EDTA around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by EDTA than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM EDTA final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction.--[[User:Kathmc\|Kathleen]]	+	*You typically want a small amount of EDTA around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by EDTA than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM EDTA final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction. [http://www.chm.bris.ac.uk/motm/edta/edtah.htm Here's] a great page I found about EDTA, including [http://www.cem.msu.edu/~cem333/EDTATable.html formation constant] (K<sub>f</sub>) values for metal-EDTA complexes.--[[User:Kathmc\|Kathleen]]

Kathmc: /* Method */

Kathmc — Wed, 04 Jan 2006 21:42:23 GMT

Method

←Older revision		Revision as of 21:42, 4 January 2006
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	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.		However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.
		+	*You typically want a small amount of EDTA around to chelate metals other than Mg. Heavier metals, like Fe, are more likely to wreck havoc on your favorite biological macromolecules and are chelated more strongly by EDTA than Mg. That said, I typically resuspend all of my oligos in TE buffer (mine is 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 μM. I use 0.5 μL of this in a 100 μL PCR reaction. This leaves me with 5 μM EDTA final, which is insignificant compared to the mM concentrations of Mg<sup>2+</sup> used in the reaction.--[[User:Kathmc\|Kathleen]]

Jason R. Kelly: /* Method */

Jason R. Kelly — Wed, 04 Jan 2006 21:21:46 GMT

Method

←Older revision		Revision as of 21:21, 4 January 2006
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	*Resuspend in [[TE buffer]], pH 8.0 at a concentration greater than 10μM.		*Resuspend in [[TE buffer]], pH 8.0 at a concentration greater than 10μM.
	*Allow to sit for 2mins, then vortex for 15s.		*Allow to sit for 2mins, then vortex for 15s.
		+
		+	However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion.

Jason R. Kelly: /* Introduction */

Jason R. Kelly — Wed, 04 Jan 2006 15:11:29 GMT

Introduction

←Older revision		Revision as of 15:11, 4 January 2006
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	==Introduction==		==Introduction==
-	When receiving oligonucleotide primers from a manufacturer such as Invitrogen, the oligos arrive dry and must be resuspended in buffer.	+	When receiving oligonucleotide primers from a manufacturer such as Invitrogen, the oligos arrive dry and must be resuspended in buffer. The proper choice of buffer will depend on the intended application of the primers, some common ones are:
		+	#Sequencing/PCR
		+	#[[Annealing Primers\|Annealing]]

	==Method==		==Method==

Barry Canton at 21:51, 12 November 2005

Barry Canton — Sat, 12 Nov 2005 21:51:19 GMT

New page

==Introduction==
When receiving oligonucleotide primers from a manufacturer such as Invitrogen, the oligos arrive dry and must be resuspended in buffer.

==Method==
Invitrogen recommends the following [http://www.invitrogen.com/content.cfm?pageid=9974 reconstitution procedure] -

*Centrifuge the tube for a few seconds to get all the DNA to the bottom of the tube.
*Resuspend in [[TE buffer]], pH 8.0 at a concentration greater than 10μM.
*Allow to sit for 2mins, then vortex for 15s.