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Forum: Sample Prep / Library Generation 11-07-2019, 10:37 AM
Replies: 1
Views: 224
Posted By microgirl123
PCR amplicon library - DNA smearing

I've been making two-step amplicon libraries for years now with very few problems. The original amplicons are tailed with partial Nextera xt adapters and then I use Phusion HF to add the rest of the...
Forum: Illumina/Solexa 10-08-2019, 09:32 AM
Replies: 2
Views: 547
Posted By microgirl123
The reason we stick with Illumina rather than...

The reason we stick with Illumina rather than going with Remi (I use them as a third-party vendor for most of our other instruments) is that Illumina is very generous with reagent replacement when...
Forum: Bioinformatics 08-06-2019, 09:21 AM
Replies: 3
Views: 558
Posted By microgirl123
The number of reads (molecules sequenced, also...

The number of reads (molecules sequenced, also called clusters) is determined by the concentration of library added. There is an optimal number the sequencing center will be aiming for depending on...
Forum: Illumina/Solexa 04-30-2019, 06:34 AM
Replies: 14
Views: 2,496
Posted By microgirl123
One possibility that would explain this is that...

One possibility that would explain this is that your phiX and library are not denaturing properly. How old is your NaOH? I always denature with NaOH, and then heat-denature my final combined...
Forum: Illumina/Solexa 04-17-2019, 12:42 PM
Replies: 2
Views: 620
Posted By microgirl123
How about using bead-based cleanup and just...

How about using bead-based cleanup and just skipping the columns entirely? I have heard of people having trouble with contamination of their PCR-grade water when they have samples with very low...
Forum: Illumina/Solexa 03-13-2019, 12:06 PM
Replies: 1
Views: 662
Posted By microgirl123
I don't have advice specific to the NovaSeq, but...

I don't have advice specific to the NovaSeq, but I had to move our MiSeq because the room it was in was slightly too hot (74deg F) so the chiller never worked optimally and the temperature always ran...
Forum: Illumina/Solexa 06-08-2018, 12:53 PM
Replies: 2
Views: 733
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: 6
Views: 2,251
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: 6
Views: 2,251
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: 2,000
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: 7,673
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: 1,778
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: 1,778
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: 7,673
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: 2,377
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: 11,654
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: 1,746
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: 1,089
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: 4,643
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: 1,422
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: 3,808
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: 1,022
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: 3,984
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: 1,960
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: 1,075
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...
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