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Old 03-20-2013, 09:14 AM   #1
jmwhitha
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Location: NC State, Raleigh, NC

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Default Removing Adapter Dimers with AMPure Beads

Can someone help?

I have adapter dimmers or some other contaminant below 200 (probably 170bp). Ultimately, my goal is to remove the contaminates and pool the libraries.

My cDNA library stocks range between 125-348ng in 15uL each, and according to Nextra Lib Validation, that's 25-67nM/ uL (1ng/uL=3nM for a 500bp avg library size). Illumina TruSeq wants me to pool 10uL of each library normalized to 10nM.

My plan is to modify the protocol I used to get my stock libraries:
3.F.1. AMPure XP Purification - http://www.epibio.com/docs/default-s...t.pdf?sfvrsn=8

First, I will transfer between 3 and 8uL of stock libary into enough RNase-Free water to make 25uL, instead of the protocols 50uL.
Second, instead of using 1 volume of AMPure XP beads, I was going to use 0.9 volumes.
Continuing through the protocol, I will do the 200uL 80% EtOH washes, air dry and resuspend the beads in 20uL. Thus I hope to get 20uL of 10nM libraries.

Does this sound like it will work?

Also, I was wondering if anyone knows why using lower volumes of AMPure XP beads removes smaller species (<200bp?) Are these retained by the beads or do they evaporate with the EtOH, maybe?

Thanks for your help!
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Old 03-20-2013, 09:32 AM   #2
ECO
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Default

Stop cross/double posting, its incredibly inconsiderate! Thanks.
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Old 03-20-2013, 10:29 AM   #3
jmwhitha
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Location: NC State, Raleigh, NC

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Posts: 105
Default Sorry, you're right

Sorry, I wasn't thinking about how people in the future might look for an answer to this question and not find it because the answer is on another post.

I was only thinking about how I could get the quickest answer by posting in multiple forums where people may find it and respond.

I will try to post a solution to each forum so that others may reap the benefits.

Let me know if I have failed to consider anything or anyone else by making similar posts in multiple forums.

Thank you!
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Old 03-20-2013, 05:08 PM   #4
jtackney
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Default

People on forums get angry at cross-posting. It's nothing new and to be considerate just delete all the other posts and keep one. People will be able to find the answer and I think this forum is the perfect place for your question

However, your question in many other forms has been asked before - do a search for SPRI/Ampure size selection and you will find many posts talking about optimal ?X beads-to-DNA to use to eliminate a certain sized fragment. You can use SPRI to keep large fragments and throw out small (one sided) or to keep something in the middle and throw out large and small (double sided). In this case, you want to remove everything below 200bp and keep the rest? That shouldn't be too difficult except if your true library is very close - 220bp for example.

One last suggestion I would make is that you really can't tell for certain how concentrated your library is going to be after SPRI - the % recovery (I have found) is too inconsistent. You would instead do the SPRI purification and then you would have to re-quantify your library. For that matter, I would not use the ng of DNA input to determine your final library concentration (unless I didn't understand what you were saying). You need to quantify your library at the end regardless.

Finally, if you want to know how SPRI works (http://core-genomics.blogspot.com/20...eads-work.html).
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Old 03-22-2013, 08:28 AM   #5
jmwhitha
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Location: NC State, Raleigh, NC

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Default Thanks for your help!

My new plan then is to first delete the other posts. Then, I will cleanup all of my sample with 0.9 beads, bioanalyze, and if it's clean, dilute to 10nM using C1 (x nM) * V1 (x uL) = C2 (10nM) * V2 (10uL). How's that?

Thanks again!
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