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Old 02-03-2013, 06:33 PM   #1
curiosity
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Post Sequence fail using PCR clean up instead of gel extraction.

Hi, I had a problem with my sequencing results. Whenever i clean up the sample using pcr clean up kit such as ampure and qiagen, in turns out to be my sequencing result fail. sometimes show short fragment and esl (early signal lost). But if i do gel extraction even the band showed bright single band, the sequence results is good. For your information, im using sanger sequencing using abi3730 analyzer. Hope somebody can give me the solution. TQVM
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Old 02-04-2013, 06:16 AM   #2
JamieHeather
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This might be a silly question, but if the gel extraction works, why not just do that?
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Old 02-05-2013, 06:04 PM   #3
curiosity
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The reason why we do clean up at the first place because the PCR product concentration is high and shows good pcr band. Normally, we proceed with gel extraction if the sample concentration is low or produce fade band. What i understand, Pcr clean up and gel extraction (same kit) used the same buffer but why the sequencing result for clean up show failed sequence?
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Old 02-06-2013, 01:20 AM   #4
JamieHeather
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So using the QIAGEN PCR purification kit doesn't work downstream, but using the gel extraction kit does? Hmm..

Well, the major difference between the two would be buffer PB I suppose - do you use the pH indicator (and does the solution remain yellow after mixing the PB with the sample)?

Are you quantitating your DNA after elution from both purifications? As in, do you get DNA from both, but there's something qualitatively different in the PCR purification versus the gel extraction?
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