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Forum: Illumina/Solexa 06-08-2018, 12:53 PM
Replies: 2
Views: 546
Posted By microgirl123
Earth Microbiome Locus Primers

Is anyone using 515F and 806R as locus primers for the Illumina two-step amplicon PCR library prep?

I have one investigator using them following the entire Earth Microbiome protocol (so adding...
Forum: Sample Prep / Library Generation 05-11-2018, 08:38 AM
Replies: 4
Views: 686
Posted By microgirl123
Well. . . it's actually an old Stratagene qPCR...

Well. . . it's actually an old Stratagene qPCR instrument that will allow you to do a single endpoint read so it's only used as a "plate" reader now.

I'm not sure how well Qubit reactions would...
Forum: Sample Prep / Library Generation 05-11-2018, 07:08 AM
Replies: 4
Views: 686
Posted By microgirl123
What are you using for quantification now? I find...

What are you using for quantification now? I find the Qubit to be time-consuming for 96 samples - instead I use the Qubit reagents, white strip tubes, and a plate reader that can handle tubes.
Forum: RNA Sequencing 11-08-2017, 08:09 AM
Replies: 5
Views: 1,580
Posted By microgirl123
You don't need to perform the bead cleanup/size...

You don't need to perform the bead cleanup/size selection to remove the larger fragments, just the smaller fragments, but you do need to remove them because they will take over a large portion of...
Forum: Illumina/Solexa 02-21-2017, 05:53 AM
Replies: 29
Views: 6,340
Posted By microgirl123
Not good :( It was an amplicon run, but the...

Not good :(

It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only...
Forum: Illumina/Solexa 02-16-2017, 06:17 AM
Replies: 11
Views: 1,430
Posted By microgirl123
If 95% of them are passing filter, then you are...

If 95% of them are passing filter, then you are definitely not overclustered. If you were overclustered your percentage passing filter would be low.

Unfortunately, the relationship between...
Forum: Illumina/Solexa 02-16-2017, 06:11 AM
Replies: 11
Views: 1,430
Posted By microgirl123
What's your percentage passing filter?

What's your percentage passing filter?
Forum: Illumina/Solexa 01-30-2017, 10:55 AM
Replies: 29
Views: 6,340
Posted By microgirl123
I just started my first 600-cycle run in over a...

I just started my first 600-cycle run in over a year so hopefully it's true! I'll try to update in a couple of days when it's finished.
Forum: Illumina/Solexa 01-27-2017, 08:25 AM
Replies: 17
Views: 2,064
Posted By microgirl123
If you Google your capitalized sequence, it comes...

If you Google your capitalized sequence, it comes up as a motif that matches "Pbx3(Homeobox)/GM12878-PBX3-ChIP-Seq/Homer." That means nothing to me, but maybe it does to you or someone else?
Forum: Illumina/Solexa 01-17-2017, 08:04 AM
Replies: 8
Views: 7,818
Posted By microgirl123
Can you post a picture of your Bioanalyzer...

Can you post a picture of your Bioanalyzer results? It sounds a bit like it might be overloaded - that can create a split peak.
Forum: Illumina/Solexa 11-09-2016, 10:46 AM
Replies: 3
Views: 1,426
Posted By microgirl123
There's no reason you can't do 250x50 with a...

There's no reason you can't do 250x50 with a 300-cycle kit. You should end up with similar Q scores to Read 1 on a 500-cycle kit, which are okay at the end (not great, but okay).
Forum: Sample Prep / Library Generation 09-22-2016, 05:52 AM
Replies: 3
Views: 852
Posted By microgirl123
Based on the Nanodrop readings, your RNA...

Based on the Nanodrop readings, your RNA concentration is pretty low, and your 260/280 is awful (especially the one that is 4.71). What are your 260/230 ratios, and what do the Nanodrop graphs look...
Forum: Illumina/Solexa 09-20-2016, 12:24 PM
Replies: 28
Views: 3,563
Posted By microgirl123
Interesting about having to pay to use all of the...

Interesting about having to pay to use all of the apps. We're going to have some unhappy customers!
Forum: Illumina/Solexa 09-12-2016, 07:27 AM
Replies: 4
Views: 1,205
Posted By microgirl123
Do you have a peak for your library also or just...

Do you have a peak for your library also or just unligated adapter/adapter dimer peaks?
Forum: Sample Prep / Library Generation 06-30-2016, 11:30 AM
Replies: 13
Views: 3,170
Posted By microgirl123
I don't get the same results from the BR and HS...

I don't get the same results from the BR and HS assays either. I think the HS assay is just not correct at the top end of the standard curve. I usually pool samples based on the HS assay and then use...
Forum: Sample Prep / Library Generation 04-08-2016, 06:54 AM
Replies: 2
Views: 819
Posted By microgirl123
I usually take a 50 ul PCR reaction and elute...

I usually take a 50 ul PCR reaction and elute into 22.5 ul. Ten ul seems extreme - you may have problems pipetting up the liquid without beads.
Forum: Sample Prep / Library Generation 03-01-2016, 07:54 AM
Replies: 11
Views: 3,197
Posted By microgirl123
I run a lot of amplicon libraries for different...

I run a lot of amplicon libraries for different labs, and most of them have problems getting rid of secondary bands with 16s amplicons. Generally, people choose to sequence everything and then...
Forum: Illumina/Solexa 02-23-2016, 06:27 AM
Replies: 4
Views: 1,665
Posted By microgirl123
This confuses a lot of our investigators too!...

This confuses a lot of our investigators too! TruSeq custom amplicon is a way to sequence many different regions of interest at once (I think it's aimed at clinical studies on things like cancer) as...
Forum: Illumina/Solexa 02-22-2016, 12:56 PM
Replies: 5
Views: 906
Posted By microgirl123
For a regular (not low-diversity) run and V3...

For a regular (not low-diversity) run and V3 reagents, I'd aim for ~1,100K/mm^2. Illumina claims you can go higher than that, but I try not to push the envelope since we're a core facility, and I'm...
Forum: Illumina/Solexa 02-22-2016, 12:40 PM
Replies: 5
Views: 906
Posted By microgirl123
The cluster density is crazy high. I'm not...

The cluster density is crazy high. I'm not surprised that nothing passed filter! Can you provide some details on what kind of library it was and how much you loaded onto the flow cell?
Forum: Sample Prep / Library Generation 02-04-2016, 11:45 AM
Replies: 2
Views: 1,321
Posted By microgirl123
To start with your ladder looks bad - your ladder...

To start with your ladder looks bad - your ladder peaks should be as high as or higher than your marker peaks at the ends. Does your ladder always look like that? If so, I'd contact Agilent and if...
Forum: Illumina/Solexa 01-29-2016, 07:32 AM
Replies: 22
Views: 4,235
Posted By microgirl123
https://my.illumina.com/MyIllumina/Bulletin/ZOCgM9...

https://my.illumina.com/MyIllumina/Bulletin/ZOCgM9hxzkmvA-vfWNrXfA/how-to-determine-a-miseq-cartridge-lot-number-from

Illumina has a bulletin about determining lot number after the fact!
Forum: Sample Prep / Library Generation 01-25-2016, 12:56 PM
Replies: 3
Views: 1,380
Posted By microgirl123
I have run a lot of amplicon samples on the...

I have run a lot of amplicon samples on the Bioanalyzer and never seen that! Since it's a high sensitivity chip, is it possible that the previous run contaminated the electrodes with something 370 bp...
Forum: Illumina/Solexa 12-23-2015, 07:01 AM
Replies: 4
Views: 1,553
Posted By microgirl123
I've had problems with high-diversity (gDNA)...

I've had problems with high-diversity (gDNA) libraries with the 600-cycle V3 kits, even with a cluster density of 1K. Read 2 just tanked after about 50 bp. We are advising people not to run them at...
Forum: Illumina/Solexa 12-09-2015, 11:35 AM
Replies: 5
Views: 3,610
Posted By microgirl123
Larger fragments don't hybridize to the MiSeq...

Larger fragments don't hybridize to the MiSeq flow cell as well as smaller ones. This can be seen when you have a small amount of adapter dimer in your (larger) libraries. The adapter dimer binds...
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