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Forum: Illumina/Solexa 01-25-2018, 07:52 AM
Replies: 23
Views: 4,529
Posted By Innovelty
Sometimes, especially on the new Illumina...

Sometimes, especially on the new Illumina flowcells, the indices get swapped around onto molecules they didn't originally belong to. With non-unique dual indices, where any combination of the index...
Forum: Illumina/Solexa 01-17-2018, 02:22 PM
Replies: 23
Views: 4,529
Posted By Innovelty
On that note, what is an unusually high...

On that note, what is an unusually high percentage of unknown reads?
Forum: Illumina/Solexa 01-17-2018, 01:08 PM
Replies: 23
Views: 4,529
Posted By Innovelty
I think it would be helpful to us to know where...

I think it would be helpful to us to know where you got your indices from, as nucacidhunter mentioned. I don't know if it's possible, but perhaps the indices from Illumina are selected because they...
Forum: Illumina/Solexa 01-17-2018, 10:47 AM
Replies: 23
Views: 4,529
Posted By Innovelty
Thanks for bumping this thread up again,...

Thanks for bumping this thread up again, systembio.

From what I know about the MiSeq chemistry (and it was the first sequencing platform I used) the unique dual indices shouldn't be a *problem*...
Forum: Sample Prep / Library Generation 09-19-2017, 10:00 AM
Replies: 0
Views: 545
Posted By Innovelty
PolyA Enrichment Failed, Now What Do I Do?

Apologies if this doubles. I tried to post before but it didn't seem to work.

I've had very good success with poly(A) / mRNA enrichment on Sera-Mag Oligo-dt beads in the past. In fact, I had...
Forum: Illumina/Solexa 08-28-2017, 08:57 AM
Replies: 23
Views: 4,529
Posted By Innovelty
Still a little confused

I'm a bit confused about how to accomplish the dual-indexing using the same index on each end. My workflow ligates an adapter, and then uses indexing primers in PCR to get flow-cell adapters and...
Forum: Vendor Forum 12-16-2014, 09:43 AM
Replies: 108
Views: 47,353
Posted By Innovelty
:p One can... provided one has access to the...

:p

One can... provided one has access to the machine, and more than a tiny pilot grant to work with. Sadly, when one works on non-model insects for non-agricultural/biomedical purposes, and one is...
Forum: Vendor Forum 12-16-2014, 05:20 AM
Replies: 108
Views: 47,353
Posted By Innovelty
Damn, thanks Brian. I woke up this morning...

Damn, thanks Brian. I woke up this morning thinking that maybe I should try a NextSeq run instead of HiSeq 2000 for this chapter of my dissertation. It seemed like I might be able to get a slightly...
Forum: Sample Prep / Library Generation 11-04-2014, 02:49 PM
Replies: 7
Views: 2,630
Posted By Innovelty
Hi Inevell, Don't sequence that library, it...

Hi Inevell,

Don't sequence that library, it isn't real.

I had some struggles with mRNA library preps in which I first got weird double peaks (a product of too many PCR cycles) and then went...
Forum: Sample Prep / Library Generation 07-09-2014, 10:43 AM
Replies: 7
Views: 3,825
Posted By Innovelty
These problems are solved. I'll attach a...

These problems are solved. I'll attach a bioanalyzer readout. Here are the relevant details regarding how I fixed it, and many thanks to Clontech Tech Support for holding my hand through it.
...
Forum: Sample Prep / Library Generation 06-25-2014, 07:20 AM
Replies: 7
Views: 3,825
Posted By Innovelty
Thanks, MU Core, after reading some more here...

Thanks, MU Core, after reading some more here that's what I figure is probably going on, as well. I tried forum user pmiguel's recommendation of heat denaturing and running on an RNA Pico chip (as...
Forum: Sample Prep / Library Generation 06-24-2014, 07:14 AM
Replies: 7
Views: 3,825
Posted By Innovelty
Clontech Stranded mRNA-Seq for Illumina: Two peaks in library?

Hi folks,

Application: poly-A RNA-Seq on a MiSeq, 2x250 pe reads.

Our desired library insert size is between 500 and 800 bp, though of course getting a size distribution that tight is tough....
Forum: Sample Prep / Library Generation 07-23-2013, 11:15 AM
Replies: 9
Views: 9,307
Posted By Innovelty
I was told that typical yield should be between 3...

I was told that typical yield should be between 3 and 8 ng/ul.

I don't know what caused this problem, because I have exactly the same problem. In my case, it was 50 ng good quality cDNA input,...
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