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Old 11-09-2012, 05:21 AM   #1
JPC
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Location: Wales

Join Date: May 2008
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Default Sureselect disappearing DNA

Hello All

We are doing the Sureselect protocol (XT all exon V4) with 3ug of DNA, after size selection we have around 1ug left for the end repair, A addition, adaptors and cleaning etc. Half of that then goes into the 6 cycles of PCR and we often have around 20uls x 30ng of DNA which is barely enough to go into the hyb.

Is anyone seeing better concentrations at this stage or is that as good as it gets?

Post hyb, after amplification we also have smallish yield (normally just enough for 1 run) is it worth doing more than the recommended 8 cycles of PCR at this stage so that we have library to come back to if we need to repeat or top up the sequence data? What concentration do you normally see after this 8 cycle PCR?

Thanks
JPC
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Old 11-12-2012, 10:06 PM   #2
ECO
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Sounds like a few problems could be going on here based on my experience.

1. ) Losses after shearing. Either you're not really putting 3ug in if you're losing that much after shearing, or your size selection is so severe that you're really losing 2/3 of your material (in addition to the purification losses). Probably related to quantitation (nanodrop) and there is a lot of degraded RNA in your gDNA and it's inflating your input concentrations. Have you ever just purified your input DNA and verified your recovery is >> 80% (Hint: if it's not, something is wrong either quantitation or technique-wise).

2.) Overall yield seems low. My rule of thumb (Assuming you amplify your whole ligation)...is that you should end library prep with (at minimum) [ER input] * (0.8)^4. This represents 80% recovery (modest) from each AMPure (post ER, post AT, post ligation, and post pre-cap PCR), and assumes the precap PCR didn't even work (ie no ligation thus no amplification). If my yields are below this level, I don't even hyb the library.

My math says that if you go into ER with 1ug, you should have 512ng at the post-ligation purification (0.8^3). Amplifying half of that (256ng) and eluting in 30ul should be 8.5ng/ul, so given your concentrations are 2.5x higher than that, it seems like that's within range for some successful amplification, but very low.

For comparison, I put 5ug of high quality genomic DNA into the Covaris, take it through library prep (including a 1.2x size cut) and pre-cap amplification (of the whole ligation) for 6 cycles, elute in 30ul, and end up in the 300-400ng/ul range (with PCR dups in the <5% range). The yield is roughly linear with reduced input material, so we'd still be in the 150-200ng/ul range with 3ug input into shearing.

Good luck! =]
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Old 11-13-2012, 01:36 AM   #3
JPC
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Location: Wales

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Many thanks for your reply. We run a gel, qubit and nanodrop for QC but there is less DNA in our sheared sample than expected so I'll try your recovery test suggestion...perhaps one of them is lying to me!

I'm a little confused by one part of your reply as you seem to suggest you're not getting a big reduction when you size select? The AB protocol (p21) states that you expect an average yield of 30% (which is what we see) you don't seem to account for that and you point out that it's pretty severe, so I'm wondering if I am missing something?

From your 5ug input DNA, with the size selection loss, you would have 5,000 * 0.3* (0.8)^4 = 30 ul @ 20.5ng/ul pre amp library (~600ng in total) then your 6 cycle PCR gives you 300-400ng/ul .... am I getting myself mixed up somewhere?

Your help is much appreciated I hope I don't sound un-grateful!

JPC

Last edited by JPC; 11-15-2012 at 06:01 AM. Reason: removed a broken link
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Old 10-24-2016, 02:25 AM   #4
sward
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Hi JPC,

I know this thread is very old but did what did you find after your recovery test? I am experiencing the same thing with my prep and can't figure out whats going on!

Many thanks !
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