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Hi, I am purifying PCR amplicons of ~650 bp for 454 titanium amplicon sequencing. I have been using 50 ul of the reaction to 40 ul of beads. I have a 50 bp ladder as control. This has worked perfectly one day and not at all the second--losing almost all of my product. The products are good clean bands, but my primers form small products under 100-150, so clean up is necessary. Does anyone have suggestions as to what I might try to get a consistently good recovery? It's the same bottle of Ampure and I have titrated it. All I can think of is that my vortexing isn't sufficient (not enough beads added) or there's some temperature or time effect. Thanks.
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Please contact me@! I would love to work with you on a more robust alternative to this product. -
Hi,
I think you may have made a miscalculation on how much Ampure XP beads to use.
The volume of Agencourt AMPure XP for a given reaction can be derived from the following
equation: (Volume of Agencourt AMPure XP per reaction) = 1.8 x (Reaction Volume).
So for 50ul PCR reaction you would use 90ul of beads. In my hands Ampure beads would not clean up small primer dimer products you describe though. These you would have to eliminate by size selection by cutting out from an agarose gel or PippinPrep.
Good luck!
Good luck!Comment
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Hi HMorrison,
I'm sorry to hear about the problems you have been encountering. I just wanted to clear up a couple of things mentioned in this thread.
Powerpuffgirl is correct that the 'standard' method using AMPure XP requires a 1.8x ratio. This will effectively clear out any products below 100bp. Small fragments should not be an issue for the kit at all, this is precisely what it is designed to do.
However, if any of your primer dimers are above 100bp (up to 150bp) then you do need to alter the ratio of AMPure XP beads added. As the cut off is not an exact thing, this will reduce the yield of the purification overall, but you should still get plenty to use in your downstream reactions. Your ratio of approximately 1.1 should get rid of fragments below 150bp effectively and still give you a decent yield of your 650bp amplicon.
This suggests there is something else going on.
Obvious question first, is the kit still in date? If the kit has expired this will decrease yields.
Secondly, are you making up the ethanol used in the washes fresh? If the ethanol is not made up fresh it will slowly take in some water from the air. This will effectively reduce the concentration of the ethanol and cause some of the product you are trying to purify to be washed off the beads.
Thirdly, how long is your final drying step? If you overdry the beads it can get very difficult to elute them off. It may require some vigourous pippetting.
Let me know if none of these apply. This is a very robust product with thousands of happy users worldwide and product quality is rarely an issue. However, if there is an issue with the kit then we would happily send you a replacement.
Good luckComment
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No further replies necessary
Hi all, my original post was 2009 and we have not been seeing problems since. If you read the original message, the problem was NOT with the ratio; we calculated that for each lot using a MW ladder. Possibly we had a bad batch of something back then, but all has been well since.Comment
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Apologies for that! I didn't think to check the date of the original question!Comment
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Sorry for that too, I thought it was a new post...Comment
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