Different band size before and after purification with Agencourt AMPure

ElenaCH · 04-02-2016, 04:45 PM

Hi everyone,

I'm doing amplification and purification of 4 different genomic regions. 3 of them are ok, but I'm struggling with my last amplicon. The amplicon length on the gel changes before and after the purification. Before purifying I have a 360 bp and after the purification I have a strong lower band of 250 and a upper band around 400 pb. It cannot be caused by a higher DNA yeld before the purification because I loaded different and small volumes on the gel to be sure about the size. It happened the same with a different primer set for the same region. Thinking about an issue with my primer set I changed it, but the problem reappeared.

Is it possible that somehow the beads interfere with the size of the amplicon? This problem never happened with other genes so I really don't understand what's going on.

Thank you for any suggestion!

Elena

nucacidhunter · 04-02-2016, 08:49 PM

Posting gel photo can give some clues on the issue. Purification cannot change the size of DNA fragment but it can cut some small fragments depending on bead/amplicon solution ratio. Amplicon before purification would be in PCR buffer with different salt concentration than after that can affect DNA migration speed affecting sizing. It is also possible that some DNA is denaturing during purification which would run as shadow band on the gel. To further investigate running on different gel%, voltage, diluting PCR before run, loading equal amplicon mass before and after purification would be useful.

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04-02-2016, 04:45 PM	#1
ElenaCH Junior Member Location: zurich Join Date: Apr 2016 Posts: 1	Different band size before and after purification with Agencourt AMPure Hi everyone, I'm doing amplification and purification of 4 different genomic regions. 3 of them are ok, but I'm struggling with my last amplicon. The amplicon length on the gel changes before and after the purification. Before purifying I have a 360 bp and after the purification I have a strong lower band of 250 and a upper band around 400 pb. It cannot be caused by a higher DNA yeld before the purification because I loaded different and small volumes on the gel to be sure about the size. It happened the same with a different primer set for the same region. Thinking about an issue with my primer set I changed it, but the problem reappeared. Is it possible that somehow the beads interfere with the size of the amplicon? This problem never happened with other genes so I really don't understand what's going on. Thank you for any suggestion! Elena

04-02-2016, 08:49 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,230	Posting gel photo can give some clues on the issue. Purification cannot change the size of DNA fragment but it can cut some small fragments depending on bead/amplicon solution ratio. Amplicon before purification would be in PCR buffer with different salt concentration than after that can affect DNA migration speed affecting sizing. It is also possible that some DNA is denaturing during purification which would run as shadow band on the gel. To further investigate running on different gel%, voltage, diluting PCR before run, loading equal amplicon mass before and after purification would be useful.