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Thread | Thread Starter | Forum | Replies | Last Post |
puzzle in fq_all2std.pl | biocc | Bioinformatics | 0 | 11-25-2010 04:33 AM |
PubMed: Targeted Massively Parallel Sequencing of Maternal Plasma DNA Permits Efficie | Newsbot! | Literature Watch | 0 | 11-17-2010 11:10 AM |
PubMed: Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation f | Newsbot! | Literature Watch | 0 | 12-23-2009 03:31 AM |
blast puzzle | anyone1985 | Bioinformatics | 1 | 09-06-2009 02:45 AM |
format puzzle | anyone1985 | Illumina/Solexa | 0 | 03-13-2009 04:29 AM |
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#1 |
Member
Location: Bethesda MD Join Date: Jun 2011
Posts: 19
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Hi All
I'm trying to isolate DNA from large volumes of plasma (anywhere from 10 to 50mL) in order to sequence it. I've been using Norgen's Circulating DNA Isolation kit but my yields have been very low so far, about 1.8ng of DNA per 5mL of plasma. Does anyone have any experience with either the Norgen kit or isolating nucleic acid from such large volumes? Thanks! |
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#2 |
Member
Location: Boston, MA Join Date: Aug 2009
Posts: 39
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I second this question. Would love to hear of any experience on the board.
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#3 |
Junior Member
Location: USA Join Date: Jul 2011
Posts: 2
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I am a scientist who has developed and clinically tested a large volume plasma sample prep system (5 - 18 ml) as part of a sepsis study. My method provides a higher yield and purity than all others we have tested.
The difference is obvious. My email address is mewert@imigene.com This is not a sales pitch. We have learned much about plasma prep as part of a US Army funded project and wish to share the discovery. |
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#4 |
--Site Admin--
Location: SF Bay Area, CA, USA Join Date: Oct 2007
Posts: 1,358
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#5 |
Junior Member
Location: USA Join Date: Jul 2011
Posts: 2
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Sorry to have breached protocol. While I am new to your forum I have been at this plasma project for some time, spent loads of cash but published none of it yet.
I just saw the request for improvements and know that we need more than the few hundred samples, which have already been tested, in order to be confident of the biochemistry we are proposing relative to nucleic acid extraction from plasma. Since I have been so busy with other matters, your forum seemed an easy way to examine potential scientific collaboration. My company is for profit however and if my request for contact is frowned upon, please accept my appology. |
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#6 |
--Site Admin--
Location: SF Bay Area, CA, USA Join Date: Oct 2007
Posts: 1,358
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I don't mind either way, just be up front about your intentions as "share" and "sell" are different things.
![]() But you're correct, typically we keep overtly commercial posts in the Vendor Forum. If you want to start a thread there specifically soliciting collaborations for your plasma DNA extraction method I will be happy to give your account permissions to post there. Thanks for understanding! |
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#7 |
Member
Location: Pittsburgh Join Date: Aug 2011
Posts: 72
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I suggest you look for the following article on pubmed."Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus." I believe this method was pioneered by Lo YM and is used in most of his papers. This is the method that we use. I would post the method but I am not sure if you get in trouble by not citing this. I don't know what your source is but we get anywhere from 0.3-1.5ng(if we are lucky) of DNA per ml of plasma. Lo says you get much more but extensive testing on our side doesn't back this up. Most of the time we don't quantify it because it is not necessary for our use but to do so you need to either do high sensitivity bioanalyzer or use qPCR methods.
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#8 |
Member
Location: Bethesda MD Join Date: Jun 2011
Posts: 19
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Thanks for the tip shawpa, I've seen a lot of papers using the QIAamp kit but have been trying to steer clear of it because of the low input volume. The addition of a speedvac helps but I'm not sure I can use that method because I have infectious samples.
I'm interested in deep sequencing the circulating DNA (illumina platform) so would like a) avoid any PCR before my libraries are prepped and b) maximize the amount of DNA I have to start with. Have you made any libraries from your samples? I've also read about differences in handling samples, some papers suggest EDTA and prompt DNA isolation to avoid sample degradation and contamination with genomic DNA as cells lyse. Some even treated with formaldehyde to stabilize DNA/prevent cell lysis. Do you have anything experience with these issues? Thanks again, WHW |
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#9 |
Member
Location: Pittsburgh Join Date: Aug 2011
Posts: 72
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Making illumina libraries is what I end up doing with my samples. Not sure what you mean by "low input volume". Do you mean that you have too much plasma that you need to process. You can reload the columns to a point. I do PCR my samples eventually according to the protocol. As for sample prep. As soon as we get the blood, we separate it and aliquot it just like the protocol says. Then store in -80 till use.
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#10 |
Member
Location: Bethesda MD Join Date: Jun 2011
Posts: 19
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low input = how much plasma you can load onto the column, just like you said. I would like to load more than 200uL in order to get the most DNA possible. anyway thanks again for the paper, those fetal DNA guys seem to know what theyre doing.
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