Fragment Size shift on Egel bye using the new TrueSeq protocol ?

[Claire1]

I am using the TruSeq protocols for RNA and DNA and I follow exactly the protocol, except I’m using an Egel (Egel: Invitrogen ‘Size Select’ 2%, Ladder: Invitrogen 100bp, 20ng/ul) before PCR instead of an Agarose Gel.
If I take my fragment size I want to use e.g. 600bp for my PCR and I run a Bioanalyzer after I see fragments around 500bp. So I have always to repeat my PCR's taking fragments around 700bp or higher to get fragments around 600bp.
This phenomenon I can see for DNA and RNA.

Anybody knows this problem?
Should I use a different Ladder?
Is the new adapter changing the migrating in the Egel?

thanks a lot for your help!

[JHU-ChIPmaniac]

Are you using the Egel specific ladders from Invitrogen? I use the 50 bp Egel ladder for my size selection of ChIP products and the isolated fragments correlate well with what we see on the bioanalyzer. I'ts possible that the regular size ladders don't run true to size on the Egels.

[Claire1]

Fragment Size shift on Egel bye using the new TrueSeq protocol ?

I'm using the 100 bp DNA Ladder from Invitrogen (Cat. 15628-050), 1ug/ul diluted to 20ng/ul.
And until now this Ladder was working fine with the old Sample Prep protocol from Illumina.
Since I switched to the new TruSeq protocol from Illumina I have problems.
Are you using the new TruSeq protocol?

Thanks for your help!

[ECO]

Is the new adapter changing the migrating in the Egel?

Almost certainly, the bioanalyzer is very sensitive to ssDNA regions. Are these anomalies pre- or post-PCR ?

[pmiguel]

ECO,
The E-gel would be run prior to PCR, the bioanalyzer after. So your take, if I understand you, is that the Y-adapters are causing migration artifacts on the E-gels. Making the fragments appear 100 bp longer than they actually are. PCR converts the Y-ends to normal double-stranded adapter ends.

I guess if the size shift is consistent Claire1 could just always extract fragments 100 bp longer than the intended size range.

We have only just started making Illumina libraries. We did see some fairly crazy size changes at various steps in the procedure during the first round of library constructions using the TruSeq kits.

For the second set of libraries we tried doing the size selection after PCR, instead of before. This was mainly because we wanted to use our new Pippin prep system and the input amount called for by its protocols could not be met without doing the PCR step first. This worked, but the yields were much lower (approaching 10x lower).

Actually, when I saw the initial result--size prior to PCR substantially larger than after PCR--I was worried that PCR was selectively amplifying a small component of the total library and that we would end up bottle-necking the library. But I don't see lots of PCR duplicates in the resulting alignments, so I think this is not what happened.

Maybe it is the E-gels that are very sensitive to the single stranded regions in pre-PCR library amplicons?

--
Phillip

[JHU-ChIPmaniac]

I'm using the 100 bp DNA Ladder from Invitrogen (Cat. 15628-050), 1ug/ul diluted to 20ng/ul.
And until now this Ladder was working fine with the old Sample Prep protocol from Illumina.
Since I switched to the new TruSeq protocol from Illumina I have problems.
Are you using the new TruSeq protocol?

Thanks for your help!

I'm not yet using the TruSeq protocol but plan on it in the future. I'll have to keep all this in mind.

[Claire1]

Even if I extract fragments 100bp higher on the Egel, I don't find the right fragment size immediately, as the ladder runs differently than the Samples.

thank you for all your answers!


Claire

[HESmith]

The TruSeq library prep protocol indicates that the new adapters cause anomalous size migration during electrophoresis either with or without ethidium bromide in the gel. It recommends using Sybr Gold staining to correct this problem.