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Forum: Sample Prep / Library Generation 05-08-2018, 10:24 AM
Replies: 13
Views: 3,169
Posted By adam.geber
DPVT, Can you elaborate on how you adjust...

DPVT,

Can you elaborate on how you adjust your qPCR measurements? Do you simply scale everything according to the discrepancy between the expected and measured molarity of the internal 200pM...
Forum: Sample Prep / Library Generation 09-14-2017, 11:08 AM
Replies: 3
Views: 1,261
Posted By adam.geber
Can you elaborate on the protocol and conditions...

Can you elaborate on the protocol and conditions (e.g. how many cycles of PCR) that you're using? Do you have a positive control reaction alongside your samples, and if so are you seeing low yield...
Forum: Sample Prep / Library Generation 09-14-2017, 11:02 AM
Replies: 2
Views: 652
Posted By adam.geber
Do you mind handling phenol in a 96-well format?...

Do you mind handling phenol in a 96-well format? If not, you could also try using Zymo's Direct-Zol 96 RNA kit -- I've been meaning to test it for tissue/cell RNA extractions for a while. If you have...
Forum: Metagenomics 08-16-2017, 09:24 AM
Replies: 4
Views: 2,183
Posted By adam.geber
Gotcha, so your control has worked well before...

Gotcha, so your control has worked well before (and is passing your standard QC) but isn't yielding anything in the context of your gradient touch-down PCR? What input mass are you using for your...
Forum: Metagenomics 08-15-2017, 10:00 AM
Replies: 3
Views: 2,152
Posted By adam.geber
Hi thermophile, Can you clarify the purpose...

Hi thermophile,

Can you clarify the purpose of spiking in the non-barcoded primers to minimize host mitochondrial contamination? Wouldn't you expect these to bind equally well to the bacterial V4...
Forum: Metagenomics 08-15-2017, 09:54 AM
Replies: 4
Views: 2,183
Posted By adam.geber
0.8x cleanups shouldn't be able to touch your...

0.8x cleanups shouldn't be able to touch your 550bp amplicons. Are you using the same polymerase recommended by Illumina? If not, then you'll likely have to do some further optimization via gradient...
Forum: Sample Prep / Library Generation 10-13-2016, 09:34 AM
Replies: 4
Views: 1,256
Posted By adam.geber
What volumes are you working with? Diluting your...

What volumes are you working with? Diluting your sample may help with clumping produced by BSA or other components of your digest.

How long are you drying your beads for? Have you tried doing...
Forum: Sample Prep / Library Generation 10-13-2016, 09:25 AM
Replies: 29
Views: 6,441
Posted By adam.geber
Those are quite decent concentrations depending...

Those are quite decent concentrations depending on your input requirements downstream -- have you decided on a prep method?

I think the best negative control would be an RNA sample split in half,...
Forum: Sample Prep / Library Generation 10-12-2016, 11:00 AM
Replies: 29
Views: 6,441
Posted By adam.geber
Your 100-150bp peak is pretty sharp, which makes...

Your 100-150bp peak is pretty sharp, which makes me think that it's not just degraded RNA and is in fact the 5S rRNA and tRNA mentioned in the RiboZero kit. Did you run a negative control for the...
Forum: Metagenomics 10-11-2016, 09:27 AM
Replies: 3
Views: 2,085
Posted By adam.geber
I think this may be one of the papers you're...

I think this may be one of the papers you're seeking out: Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing...
Forum: Sample Prep / Library Generation 08-24-2016, 08:34 AM
Replies: 4
Views: 1,056
Posted By adam.geber
yuehuang, would you mind sharing your findings...

yuehuang, would you mind sharing your findings here when appropriate? I use a different method to make 16S libraries but have experienced similar sizing discrepancies in the past so I'm interested to...
Forum: Sample Prep / Library Generation 07-12-2016, 05:38 PM
Replies: 10
Views: 1,445
Posted By adam.geber
1) The D5000 assay has 10% sizing accuracy,...

1) The D5000 assay has 10% sizing accuracy, which is within the range of the fragment sizes you're seeing. Have you seen a size discrepancy for your libraries across multiple preps?

2) PCR bias...
Forum: Sample Prep / Library Generation 03-21-2016, 03:11 PM
Replies: 3
Views: 2,194
Posted By adam.geber
Nextera XT requires input DNA of fairly low...

Nextera XT requires input DNA of fairly low concentration (0.2ng/uL). What platforms are you using to measure your samples' concentration? Are you using QIAxcel readings to make your dilutions before...
Forum: Sample Prep / Library Generation 03-21-2016, 03:00 PM
Replies: 4
Views: 2,035
Posted By adam.geber
Can you describe your current protocol with more...

Can you describe your current protocol with more detail? Unless you're starting from influenza culture samples you won't get good coverage of your genome without further enrichment for viral nucleic...
Forum: Sample Prep / Library Generation 03-02-2016, 12:06 PM
Replies: 11
Views: 3,194
Posted By adam.geber
Out of curiosity, for folks using the 515f/806r...

Out of curiosity, for folks using the 515f/806r Caporaso primers are you having trouble getting your libraries to cluster despite seeding at the expected concentration? I'm currently doing a...
Forum: Sample Prep / Library Generation 02-22-2016, 10:09 AM
Replies: 1
Views: 722
Posted By adam.geber
Hi Jakob, What method are you using to...

Hi Jakob,

What method are you using to extract your RNA and how are you storing it (i.e. buffer, conditions, etc.)? If your extraction is yielding high-purity, intact RNA and both sets are stored...
Forum: Sample Prep / Library Generation 02-10-2016, 03:36 PM
Replies: 0
Views: 852
Posted By adam.geber
MICROBEnrich 28S removal

I'm using Life Technologies' MICROBEnrich and MICROBExpress kits in conjunction to deplete prokaryotic/eukaryotic rRNAs and polyadenylated transcripts through capture hybridization. I'm consistently...
Forum: Sample Prep / Library Generation 01-31-2016, 12:44 PM
Replies: 7
Views: 1,830
Posted By adam.geber
The ideal profile for your samples depends almost...

The ideal profile for your samples depends almost entirely on what you're trying to sequence and to what end. For instance, with whole genome shotgun sequencing you don't need to worry too much about...
Forum: Sample Prep / Library Generation 12-12-2015, 01:48 PM
Replies: 10
Views: 2,801
Posted By adam.geber
marymac, one obvious question: which version of...

marymac, one obvious question: which version of the RNeasy kit were you using? I find that the RNeasy Micro kit worked far better at retaining small amounts (<100ng) of RNA than the Mini version.
...
Forum: Sample Prep / Library Generation 12-10-2015, 05:13 PM
Replies: 10
Views: 2,801
Posted By adam.geber
Exactly. One of my collaborators uses Terminator...

Exactly. One of my collaborators uses Terminator exonuclease...
Forum: Sample Prep / Library Generation 12-10-2015, 11:48 AM
Replies: 10
Views: 2,801
Posted By adam.geber
This certainly wouldn't be the first time that...

This certainly wouldn't be the first time that I've seen RNAlater complicate extractions downstream -- it's certainly possible that it's not penetrating the cuticles. One of the best options with...
Forum: Metagenomics 12-10-2015, 10:26 AM
Replies: 5
Views: 1,930
Posted By adam.geber
Can you describe your DNase treatment more...

Can you describe your DNase treatment more thoroughly? Also, what size filters were you using? Viral-like particle enrichment is quite difficult and needs to be tailored to each sample type in order...
Forum: Sample Prep / Library Generation 12-10-2015, 10:05 AM
Replies: 10
Views: 2,801
Posted By adam.geber
Hi Mary, 1 & 2 are not inconsistent with...

Hi Mary,

1 & 2 are not inconsistent with each other -- the Nanodrop is known to heavily overestimate concentration, especially in the presence of contaminants. Have you checked your samples for...
Forum: Sample Prep / Library Generation 12-09-2015, 01:40 PM
Replies: 2
Views: 2,286
Posted By adam.geber
Hi evobio, One method I've started using to...

Hi evobio,

One method I've started using to avoid residual phenol contamination is performing a standard Trizol extraction up to phase separation followed by passing the aqueous phase through a...
Forum: Sample Prep / Library Generation 12-09-2015, 09:38 AM
Replies: 21
Views: 3,111
Posted By adam.geber
Definitely a good idea to store at a higher...

Definitely a good idea to store at a higher concentration than needed for submission. For reference, you'll need to redilute your 151nM library to 10nM with e.g. 5uL of your library in 70.5uL of...
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