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Old 08-01-2016, 06:42 AM   #1
ARDISSON
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Location: Montpellier

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Default Long range PCR / purication AMpure XP

Hi,
I make a librairies around 6Kb for sequencing.
I shear and size DNA and I add adaptator by ligation. After each step, I do a purification with 1x SPRI AMPure XP beads without particular problem.
After long range PCR, I would purifiy with 1x SPRI AMPure XP beads. But something stop an optimal purification during the elution.
I tested 2 enzyme: Long Amp by NEB and Prime STAR by TAKARA but I have the same problem. The Suppliers tell me there are no incompatibilities between the PCR buffer and AMpure XP beads.

Does anyone ever had this problem ?
Thanks
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Old 08-01-2016, 11:17 AM   #2
nucacidhunter
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Could you give more details on the problem, for instance low yield or wrong size. For PCR clean up there is option of using columns as well if throughput is not high.
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Old 08-02-2016, 05:30 AM   #3
ARDISSON
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Hi,
the problem is not for instance low yield or wrong size, I think is a incompatibility between reaction product PCR and SPRI AMPure XP beads, because during water elution, beads make a strange heap. When I put DNA elution on Agilent 2100 Analyser or Agarose gel, I see very strange think like smear...
thank you in advance for your help
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Old 08-02-2016, 02:47 PM   #4
nucacidhunter
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I have not had any issue cleaning PrimeSTAR reactions with beads. Things to consider:
1- Plasticware
2- Residual ethanol
3- AMPure itself
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Old 08-03-2016, 12:02 AM   #5
ARDISSON
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I'll start paying attention to these 3 points.
Thanks
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