Forum: Sample Prep / Library Generation
05-08-2018, 10:24 AM
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Replies: 13
Views: 4,784
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...
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Forum: Sample Prep / Library Generation
09-14-2017, 11:08 AM
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Replies: 3
Views: 1,949
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Forum: Sample Prep / Library Generation
09-14-2017, 11:02 AM
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Replies: 2
Views: 1,058
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Forum: Metagenomics
08-16-2017, 09:24 AM
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Replies: 4
Views: 4,966
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...
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Forum: Metagenomics
08-15-2017, 10:00 AM
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Replies: 3
Views: 4,473
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...
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Forum: Metagenomics
08-15-2017, 09:54 AM
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Replies: 4
Views: 4,966
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...
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Forum: Sample Prep / Library Generation
10-13-2016, 09:34 AM
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Replies: 4
Views: 2,430
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Forum: Sample Prep / Library Generation
10-13-2016, 09:25 AM
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Replies: 29
Views: 8,062
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Forum: Sample Prep / Library Generation
10-12-2016, 11:00 AM
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Replies: 29
Views: 8,062
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Forum: Metagenomics
10-11-2016, 09:27 AM
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Replies: 3
Views: 2,888
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Forum: Sample Prep / Library Generation
08-24-2016, 08:34 AM
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Replies: 4
Views: 1,843
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...
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Forum: Sample Prep / Library Generation
07-12-2016, 05:38 PM
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Replies: 10
Views: 2,542
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...
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Forum: Sample Prep / Library Generation
03-21-2016, 03:11 PM
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Replies: 3
Views: 3,219
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...
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Forum: Sample Prep / Library Generation
03-21-2016, 03:00 PM
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Replies: 4
Views: 2,362
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Forum: Sample Prep / Library Generation
03-02-2016, 12:06 PM
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Replies: 11
Views: 4,955
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Forum: Sample Prep / Library Generation
02-22-2016, 10:09 AM
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Replies: 1
Views: 1,032
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...
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Forum: Sample Prep / Library Generation
02-10-2016, 03:36 PM
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Replies: 0
Views: 1,476
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...
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Forum: Sample Prep / Library Generation
01-31-2016, 12:44 PM
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Replies: 7
Views: 2,988
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Forum: Sample Prep / Library Generation
12-12-2015, 01:48 PM
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Replies: 10
Views: 4,257
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.
...
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Forum: Sample Prep / Library Generation
12-10-2015, 05:13 PM
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Replies: 10
Views: 4,257
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Forum: Sample Prep / Library Generation
12-10-2015, 11:48 AM
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Replies: 10
Views: 4,257
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...
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Forum: Metagenomics
12-10-2015, 10:26 AM
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Replies: 5
Views: 2,678
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...
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Forum: Sample Prep / Library Generation
12-10-2015, 10:05 AM
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Replies: 10
Views: 4,257
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...
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Forum: Sample Prep / Library Generation
12-09-2015, 01:40 PM
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Replies: 2
Views: 3,457
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...
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Forum: Sample Prep / Library Generation
12-09-2015, 09:38 AM
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Replies: 21
Views: 4,193
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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