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Forum: Sample Prep / Library Generation 02-06-2020, 09:22 PM
Replies: 1
Views: 409
Posted By luc
I believe you can use standard barcoded Nextera...

I believe you can use standard barcoded Nextera primers instead. Preferentially UDI dual indexed versions of the Nextera primers.
Simple desalted oligos will be fine indeed.
Forum: Sample Prep / Library Generation 02-06-2020, 09:09 PM
Replies: 21
Views: 5,571
Posted By luc
Thanks. No manual available yet. List price is...

Thanks. No manual available yet. List price is $82 per reaction.
Forum: Sample Prep / Library Generation 02-05-2020, 07:00 PM
Replies: 21
Views: 5,571
Posted By luc
According to rumors, RiboZero will make a...

According to rumors, RiboZero will make a comeback. Not exactly the same products though; it will use a new chemistry.
Forum: Sample Prep / Library Generation 02-05-2020, 06:53 PM
Replies: 8
Views: 1,807
Posted By luc
Thanks Adam, this image is very helpful!

Thanks Adam,
this image is very helpful!
Forum: General 02-04-2020, 11:21 PM
Replies: 1
Views: 451
Posted By luc
To me the data you mentioned are no big reason to...

To me the data you mentioned are no big reason to worry. They gave you more data for the first sample, but duplicates are certianly higher than expected.
The biases visible for the first 16 nt in...
Forum: Sample Prep / Library Generation 02-04-2020, 11:09 PM
Replies: 8
Views: 1,807
Posted By luc
Hi Adam, the promised attached image seems to...

Hi Adam,
the promised attached image seems to be missing.
Forum: Bioinformatics 01-26-2020, 11:05 PM
Replies: 2
Views: 405
Posted By luc
Could you describe where your barcodes/indices...

Could you describe where your barcodes/indices are located in the Illumina reads?
In the index reads (i5 and i7) or in-line with the forward and reverse sequence reads?
Forum: Core Facilities 12-02-2019, 06:20 PM
Replies: 2
Views: 1,299
Posted By luc
What assay do you want or run with the converted...

What assay do you want or run with the converted sample?
Forum: Sample Prep / Library Generation 11-19-2019, 08:34 AM
Replies: 1
Views: 752
Posted By luc
Best ask Nugen. Likely Nugen is using the Truseq...

Best ask Nugen. Likely Nugen is using the Truseq design for their kits, not Nextera sequences.
Forum: Introductions 11-07-2019, 05:29 PM
Replies: 1
Views: 710
Posted By luc
Welcome Saba! ...

Welcome Saba!
http://kellis.blogs.ccps.us/files/2016/08/cropped-cropped-scientist-clipart-science-clip-art-school1.png
Forum: Sample Prep / Library Generation 11-07-2019, 05:24 PM
Replies: 2
Views: 1,025
Posted By luc
Not a good explanation, nevertheless: perhaps...

Not a good explanation, nevertheless: perhaps your stock of index primers is degrading or got contaminated? You might want to order a few fresh oligos and compare?
Forum: Illumina/Solexa 11-05-2019, 09:02 AM
Replies: 18
Views: 1,550
Posted By luc
This is using custom sequencing primers? The...

This is using custom sequencing primers?

The heating and cooling elements are not calibrated exactly the same for all MiSeqs (by Illumina). Some risky custom sequencing primer designs can work...
Forum: Introductions 11-04-2019, 08:18 PM
Replies: 1
Views: 705
Posted By luc
Well ........ welcome Tom! ...

Well ........ welcome Tom!

https://images-na.ssl-images-amazon.com/images/S/sgp-catalog-images/region_US/wb-883316329542-Full-Image_GalleryBackground-en-US-1484348613962._SX1080_.jpg
Forum: Sample Prep / Library Generation 10-30-2019, 04:34 PM
Replies: 2
Views: 1,028
Posted By luc
Yep, bead cleanups will never be perfect. You can...

Yep, bead cleanups will never be perfect. You can only enrich for molecules of the desired size range.
Many protocols suggest two rounds of bead cleanups likey to safeguard for less than optimal...
Forum: Sample Prep / Library Generation 10-08-2019, 06:18 PM
Replies: 10
Views: 1,368
Posted By luc
It will. Who says molecular...

It will.

Who says molecular physics/chemistry is easy? These signals are likely due to the nature of dyes. Likely the HS-DNA dye will mostly bind to dsDNA to become fluorescent, but also to...
Forum: Sample Prep / Library Generation 10-03-2019, 12:08 PM
Replies: 10
Views: 1,368
Posted By luc
I guess your RNA coul actually be clean, DNA-free.

I guess your RNA coul actually be clean, DNA-free.
Forum: Sample Prep / Library Generation 10-03-2019, 09:21 AM
Replies: 10
Views: 1,368
Posted By luc
Qubit DNA assays will also measure Doublestranded...

Qubit DNA assays will also measure Doublestranded RNA, e.g. ds parts of rRNA.
Do you see any signs of DNA on the Bioanalyzer?
Forum: Sample Prep / Library Generation 09-11-2019, 05:19 PM
Replies: 1
Views: 1,193
Posted By luc
I am not aware of such a protocol. Best sequence...

I am not aware of such a protocol. Best sequence the library to low coverage as a spike-in into another run first - using different barcodes obviously.
Forum: Sample Prep / Library Generation 09-11-2019, 05:14 PM
Replies: 5
Views: 1,364
Posted By luc
Very, very likely your libraries will be...

Very, very likely your libraries will be perfectly fine. 45C should not be a problem, even in pure H2O. This is my guess, though.
Forum: Sample Prep / Library Generation 09-09-2019, 04:37 PM
Replies: 5
Views: 1,364
Posted By luc
I believe this recommendation usually applies to...

I believe this recommendation usually applies to RNA samples - which are more heat sensitive.

Depending on the buffer (some labs might use be pure H2O) and the lengths of fragments, using low...
Forum: Sample Prep / Library Generation 09-05-2019, 10:14 AM
Replies: 1
Views: 1,158
Posted By luc
What type of size selection did you use? I...

What type of size selection did you use?
I guess, these results are likely due to the lack of size-selection and low inputs? since you were working low integrity/quality RNA samples from fixed...
Forum: Sample Prep / Library Generation 09-03-2019, 04:29 PM
Replies: 1
Views: 875
Posted By luc
I had not seen these before. The idea looks very...

I had not seen these before. The idea looks very interesting.

I would assume the XNA clamps are added to the PCR amplification and that they are universal for the TruSeq adapter sequences. Best...
Forum: Sample Prep / Library Generation 08-15-2019, 05:53 PM
Replies: 2
Views: 918
Posted By luc
What type of libraries are you generating? How...

What type of libraries are you generating? How are you measuring concentrations?

I would suggest to avoid Phusion for complex libraries. It is fine for amplicon sequencing. Phusion has been shown...
Forum: General 08-15-2019, 05:13 PM
Replies: 1
Views: 2,227
Posted By luc
It is not necessary to bring all libraries to the...

It is not necessary to bring all libraries to the same concentration before pooling - although many labs do it that way.
You could also pool equal amounts of each library (e.g. same number of...
Forum: Sample Prep / Library Generation 08-13-2019, 09:32 AM
Replies: 1
Views: 823
Posted By luc
In our experience Quantseq seems to perform...

In our experience Quantseq seems to perform better than other 3' tag-seq methods that rely on tagmentation for adding the 5' adapter. We do not have hard data, though.
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