loss of library DNA in the last purification, need help

Anjani · 11-18-2011, 02:32 AM

Hi,
I'm doing library preparation for SOLiD 5500. I have no problem during the process (size selection, dA tail, adaptor ligation etc), until the purification step after amplifying the library. We have no DNA left after purification

. I used Agencourt AMPure beads for purification. Is anybody has the same problem and know is/are the cause(s), and know how to solve it? Our group has the same problem before.
Many thanks

jinpingzhang · 09-06-2012, 09:50 PM

Hi, Anjani,
i do not know what kind of your library, i try to contribute some minds.Ok, Agencourt AMPure beads is excellent product for purefication for DNA and RNA, they have RNA Clean beads for clean RNA specificlly. even i already try single product, it is work very good.
i just gess they use PEG8000 and NaCl as bead suspention buffer. maybe the concentration is 13%PEG/1.25M NaCI/10mM MgCI2, we dont care that. if you dont like that, try these way:62% ethanol in final concentration, plus 500mM NH4OAc in final concentration. for example: if your DNA/RNA solution is 100ul, add 100% Ethanol 221ul and 36ul 5M NH4OAc to total 357ul. on magnetic rack 1min, discard original bead supernatant, suspend in your made solution. at room temp mix well and incubate less 5minutes, then set in a magnetic rack, discard supernatant, dry to near crack pellet, dont over dry!! elute your H2O or TE.
remember please, 55%-62% ethanol will give your gold range for purefication process, depending your experiment request.
good luck

09-13-2012, 05:39 AM

This has also happened to me once during the final size selection of the library end product. I must have not been careful with getting ever microliter right and I had no library of the expected size in the end. So I just repeated the washing and cut off on the same beads being extra careful (hoping my product was still sticking to them) and managed to successfully elute the product from the beads.

elenstea · 06-27-2014, 12:52 AM

We had the same problem several time, we stopped using beads, now using spin columns and we have no problem

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11-18-2011, 02:32 AM	#1
Anjani Junior Member Location: Lyon, France Join Date: Nov 2011 Posts: 1	loss of library DNA in the last purification, need help Hi, I'm doing library preparation for SOLiD 5500. I have no problem during the process (size selection, dA tail, adaptor ligation etc), until the purification step after amplifying the library. We have no DNA left after purification . I used Agencourt AMPure beads for purification. Is anybody has the same problem and know is/are the cause(s), and know how to solve it? Our group has the same problem before. Many thanks

09-06-2012, 09:50 PM	#2
jinpingzhang Member Location: Houston Join Date: Mar 2012 Posts: 10	Hi, Anjani, i do not know what kind of your library, i try to contribute some minds.Ok, Agencourt AMPure beads is excellent product for purefication for DNA and RNA, they have RNA Clean beads for clean RNA specificlly. even i already try single product, it is work very good. i just gess they use PEG8000 and NaCl as bead suspention buffer. maybe the concentration is 13%PEG/1.25M NaCI/10mM MgCI2, we dont care that. if you dont like that, try these way:62% ethanol in final concentration, plus 500mM NH4OAc in final concentration. for example: if your DNA/RNA solution is 100ul, add 100% Ethanol 221ul and 36ul 5M NH4OAc to total 357ul. on magnetic rack 1min, discard original bead supernatant, suspend in your made solution. at room temp mix well and incubate less 5minutes, then set in a magnetic rack, discard supernatant, dry to near crack pellet, dont over dry!! elute your H2O or TE. remember please, 55%-62% ethanol will give your gold range for purefication process, depending your experiment request. good luck

09-13-2012, 05:39 AM	#3
lorendarith Guest Posts: n/a	This has also happened to me once during the final size selection of the library end product. I must have not been careful with getting ever microliter right and I had no library of the expected size in the end. So I just repeated the washing and cut off on the same beads being extra careful (hoping my product was still sticking to them) and managed to successfully elute the product from the beads.

06-27-2014, 12:52 AM	#4
elenstea Junior Member Location: Greece Join Date: May 2014 Posts: 7	We had the same problem several time, we stopped using beads, now using spin columns and we have no problem