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Forum: Sample Prep / Library Generation 06-19-2019, 04:27 PM
Replies: 2
Views: 979
Posted By luc
We have not tried this protocol. One question is:...

We have not tried this protocol. One question is: are your cells in a buffer very low in metals like Mg++ ? The protocol suggests a 1:500 dilution of Phusion HF buffer (presumably 1:500 of a 1x...
Forum: Illumina/Solexa 06-15-2019, 10:10 AM
Replies: 2
Views: 376
Posted By luc
No compatibility problems at all.

No compatibility problems at all.
Forum: Sample Prep / Library Generation 06-12-2019, 04:51 PM
Replies: 9
Views: 4,635
Posted By luc
Covaris does now sell plastic plates and strips...

Covaris does now sell plastic plates and strips for shearing on their very latest sonicators:
https://covaris.com/products/afa-tubes-and-vials/afa-tube/
They are a tiny bit cheaper.



You...
Forum: Bioinformatics 05-28-2019, 09:23 AM
Replies: 2
Views: 380
Posted By luc
??? UMIs have been used since at least...

???

UMIs have been used since at least 2013, then sequenced on an old GAII:
"Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide...
Forum: Illumina/Solexa 05-24-2019, 05:25 PM
Replies: 11
Views: 700
Posted By luc
Illumina did upgrade the HS2500 sequencers of a...

Illumina did upgrade the HS2500 sequencers of a few selected companies to V4 chemistry compatibility - overall very few such compatible machines did exist. With this chemistry however, the HS2500 is...
Forum: Illumina/Solexa 05-24-2019, 12:51 PM
Replies: 11
Views: 700
Posted By luc
Very likely yes, since Illumina is manipulating...

Very likely yes, since Illumina is manipulating the prices to their liking. HS4K reagent costs did increase by 6% this year (everything else by 3%); the costs for the small NovaSeq flowcells OTOH...
Forum: General 05-23-2019, 06:30 PM
Replies: 6
Views: 496
Posted By luc
You will need a magnetic bead separation plate to...

You will need a magnetic bead separation plate to use the beads for cleanups in 96-well PCR plates.

Zymo has DNA clean & concentrate kits in plate format (silica columns). You will require a...
Forum: Illumina/Solexa 05-22-2019, 04:52 PM
Replies: 3
Views: 419
Posted By luc
I would check out the SMART-SEQ2 protocol. They...

I would check out the SMART-SEQ2 protocol. They did homebrew something similar. I vaguely remember SDS to denature the enzyme.
Forum: Illumina/Solexa 05-22-2019, 04:38 PM
Replies: 11
Views: 700
Posted By luc
I guess it is not the throughput nor the lifespan...

I guess it is not the throughput nor the lifespan of the instruments that kills the older sequencers; it is mostly the comparatively high costs per base. The exception are the MiSeqs because Illumina...
Forum: General 05-21-2019, 08:43 PM
Replies: 3
Views: 457
Posted By luc
The presence of a prominent short fragment band...

The presence of a prominent short fragment band indicates that degradation is not the problem. Bacterial RNA-seq obviously will not make use of poly-A enrichment and thus is not very sensitive to...
Forum: Sample Prep / Library Generation 05-14-2019, 07:29 PM
Replies: 6
Views: 506
Posted By luc
I do not believe that the bead surface is...

I do not believe that the bead surface is strongly limiting the DNA amounts that can be precipitated onto it - at least not when using standard Ampure (PEG/NaCl) buffer.
It is worth a try.
Forum: Sample Prep / Library Generation 05-10-2019, 06:20 AM
Replies: 2
Views: 476
Posted By luc
What QC information do you have for the libraries...

What QC information do you have for the libraries before pooling and capture?
After sequencing, how is the read number within the pools?
Forum: Introductions 05-07-2019, 06:54 AM
Replies: 2
Views: 374
Posted By luc
Welcome to the forum! But better ask such...

Welcome to the forum!
But better ask such questions in the library generation forum.
That said I can't imagine that this would generate usable data - I assume the fixation is too strong in FFPE...
Forum: Illumina/Solexa 05-06-2019, 04:58 PM
Replies: 10
Views: 971
Posted By luc
I am just speculating. AFAIU, the remaining...

I am just speculating. AFAIU, the remaining oligos will be kicked off by nick-translation/strand-displacement in addition to the gap filling; I assume there is no ligation happening. (Perhaps the...
Forum: Illumina/Solexa 04-26-2019, 09:54 AM
Replies: 3
Views: 1,187
Posted By luc
Every library molecule and every strand has both...

Every library molecule and every strand has both the P5 and P7 sequences.
Only one of the ends hybridizes initially, because of the direction/complementarity of the flowcell oligos.
Forum: Illumina/Solexa 04-22-2019, 02:12 PM
Replies: 2
Views: 408
Posted By luc
PCR inhibitors?

PCR inhibitors?
Forum: Sample Prep / Library Generation 04-19-2019, 04:45 PM
Replies: 6
Views: 1,016
Posted By luc
Sorry, I have no clue what could have gone wrong....

Sorry, I have no clue what could have gone wrong. We do see significantly higher yields with the same Kapa kit using Biooscientific adapters after 7 PCR cycles.
Did you run a two-sided size...
Forum: Illumina/Solexa 04-16-2019, 06:19 PM
Replies: 2
Views: 447
Posted By luc
PCR cleanup should be comparatively "easy" and...

PCR cleanup should be comparatively "easy" and all the usual manufacturers should have good options.
Qiagen QIAquick PCR or Zymo DNA clean would be some we have used.
Forum: Sample Prep / Library Generation 04-12-2019, 08:17 PM
Replies: 6
Views: 1,016
Posted By luc
How many PCR cycles did you run? Do the libraries...

How many PCR cycles did you run? Do the libraries have the expected length?
Forum: Sample Prep / Library Generation 04-10-2019, 08:09 AM
Replies: 18
Views: 1,203
Posted By luc
Hi KB*, thanks, the Nuclease treatment...

Hi KB*,

thanks, the Nuclease treatment explains the shorter than usual fragments.
You really became creative with the protocol. I hope it works out.

I am not worried about a library degrading...
Forum: Illumina/Solexa 04-08-2019, 06:39 PM
Replies: 5
Views: 436
Posted By luc
Sorry, I do not fully understand what type of...

Sorry, I do not fully understand what type of information you have and what you are looking to retrieve.
Do you see the index read sequences in the read headers?
Forum: Sample Prep / Library Generation 04-08-2019, 09:51 AM
Replies: 18
Views: 1,203
Posted By luc
Which sonicator did you use for your chromatin? ...

Which sonicator did you use for your chromatin? Usually, it is very difficult to achieve such small inserts.

The seq facility is certainly correct that the size distribution is not optimal...
Forum: Sample Prep / Library Generation 04-04-2019, 12:00 PM
Replies: 4
Views: 511
Posted By luc
You misunderstood me: Long fragments will...

You misunderstood me: Long fragments will require a longer time to elute/dissolve from the beads compared to shorter fragments.
Forum: Sample Prep / Library Generation 04-03-2019, 07:23 PM
Replies: 18
Views: 1,203
Posted By luc
This is ATAC-seq? The proportion of longer...

This is ATAC-seq?
The proportion of longer fragments is relatively small. In our experience libraries like these can be sequenced without issues.
I do not believe the magnets are to blame - likely...
Forum: Sample Prep / Library Generation 04-03-2019, 06:19 PM
Replies: 18
Views: 1,203
Posted By luc
The attached images are too small for me to read...

The attached images are too small for me to read the axis units. However, the libraries seem to look perfectly fine for me.
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