Ampure XP Hack

sjcire · 11-29-2010, 04:34 PM

For Chip-Seq library generation, in the size select step, I have had many problems using agarose; low resolution of short nucleotides and low % recovery using the min-elute columns. I prefer to use a TBE acrylamide gel. In order to do so, acrylamide gels electro-elution was employed, see Sambrook's molecular cloning. The problem with eletro-eluiton, in my hands, is that in the end I'm left with > 350ul of a dilute DNA solution. To use with Ampure beads, following the standard protocol, > 630ul of Ampure was required. This makes it an expensive step. I therefore have made an attempt to hack the kit. Please find attached the protocol I use and its companion excel spreadsheet. Archive.zip

I have used this protocol to purify 1ml 1ng/ul DNA sample with 10ul Ampure beads. I achieved ~40% recovery. This may save you 1 EtOH precipitation or save some of your Ampure XP.
I should add that standard column purification is easier and better suited for this type of application. However, Ampure XP has better size exclusion properties(removal of primers and adapter dimers). These properties make it well suited for ChIP-Seq library generation, in my opinion.

Please let me know about typos and mistakes.

mikmz · 07-02-2012, 07:16 AM

Thank you for this information, it was very helpful.

Do you know if the Ampure beads are silica-coated and functionalised with -COOH? If so, is the buffer that you have described a -COOH-specific buffer?

I have heard that the patent for this buffer has run out.

Also, can you explain how you came to calculate the correct volumes of PEG and NaCl in the buffer, so that the correct final concentration of each is reached?

Thanks.

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11-29-2010, 04:34 PM	#1
sjcire Junior Member Location: Corvallis OR Join Date: Nov 2010 Posts: 4	Ampure XP Hack For Chip-Seq library generation, in the size select step, I have had many problems using agarose; low resolution of short nucleotides and low % recovery using the min-elute columns. I prefer to use a TBE acrylamide gel. In order to do so, acrylamide gels electro-elution was employed, see Sambrook's molecular cloning. The problem with eletro-eluiton, in my hands, is that in the end I'm left with > 350ul of a dilute DNA solution. To use with Ampure beads, following the standard protocol, > 630ul of Ampure was required. This makes it an expensive step. I therefore have made an attempt to hack the kit. Please find attached the protocol I use and its companion excel spreadsheet. Archive.zip I have used this protocol to purify 1ml 1ng/ul DNA sample with 10ul Ampure beads. I achieved ~40% recovery. This may save you 1 EtOH precipitation or save some of your Ampure XP. I should add that standard column purification is easier and better suited for this type of application. However, Ampure XP has better size exclusion properties(removal of primers and adapter dimers). These properties make it well suited for ChIP-Seq library generation, in my opinion. Please let me know about typos and mistakes. Last edited by sjcire; 11-29-2010 at 04:38 PM.

07-02-2012, 07:16 AM	#2
mikmz Junior Member Location: Ireland Join Date: Sep 2011 Posts: 2	Thank you for this information, it was very helpful. Do you know if the Ampure beads are silica-coated and functionalised with -COOH? If so, is the buffer that you have described a -COOH-specific buffer? I have heard that the patent for this buffer has run out. Also, can you explain how you came to calculate the correct volumes of PEG and NaCl in the buffer, so that the correct final concentration of each is reached? Thanks. Last edited by mikmz; 07-03-2012 at 01:13 AM.