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Old 11-06-2013, 05:01 AM   #1
WhiteSeal
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Location: Netherlands

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Question TruSeq Nano DNA sample prep

Hi Everyone,

I have done a number of TruSeq DNA preps, but now that it will be discontinued we have been looking into the TruSeq Nano prep. I have been reading the guide, but I found in the 'romoval of large DNA fragments' step things are confusing.

I see that 125ul needs to be transferred into a new plate (twice, thus that each plate will contain 250 ul)... ? So is this 1 plate with 250ul or two plates with each 125ul?

This continues with the removal of small fragments where 138ul and 276ul are also used as if it is the same? Really confusing and I don't see how this passed Illumina's check before it went out.

Did anyone come across this as well and how did you continue the prep?

With regards,

Dirk van essen
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Old 11-06-2013, 06:11 AM   #2
SS00
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Hi Dirk,

I have come across this, but didn't find it confusing. I think the reason Illumina asks you to remove the supernatant in two steps is because a 1ml pipette is too big/clumsy to handle without touching the beads. Let me see if I can explain it how I understood it:

To Remove Large Fragments You:
Add 160ul diluted bead mixture to 100ul End Repair Product. Your total volume is 260ul.
The beads contain the large fragments and the supernantant smaller.

To Isolate the Supernatant for the 2nd Sided Size Selection (Remove Small Fragments) You need to remove 250ul of supernatant into a new plate (Leaving 10ul behind to avoid disturbing the beads and taking along large fragments you don't want).
However, instead of Illumina advising you to do this at once with a large 1000ul pipette tip and risk you disturbing the beads or not being accurate with the volume they ask you to do this with a smaller 200ul pippette. Because of the volume limitation of a 200ul pipette they ask you to do this twice.
So x 125ul goes into the SAME well in the sample plate twice to total 250ul of supernatant.

Same goes for the remove of small fragments after adding 30ul beads to the 250ul supernantant you just isolated - total 280ul. Small fragments are in supernatant, target fragments on beads. In this step you need to remove most of the supernatant and then proceed to the EtOH wash steps. Instead of asking you to remove most of the supernant at once (with a 1000ul pipette tip) Illumina splits the volume in two - so you remove 138 twice, removing a total of 276ul supernant and leaving behind a negligeable 4ul to avoid disturbing the beads.

Does that make any more sense?
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Old 11-06-2013, 06:54 AM   #3
WhiteSeal
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Ah, that does make sense. I was confused by the 'Each CEP plate' line in the protocol, suggesting that there are two plates.

Maybe they should just have changed the line to 'Use a 200 μl single channel to transfer, do not discard, the supernatant of 250ul'.

Thanks for clearing that up for me.

Dirk
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