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Forum: Sample Prep / Library Generation 05-28-2019, 01:45 AM
Replies: 4
Views: 1,121
Posted By JBKri
I have sometimes used Ampure beads that were 1-3...

I have sometimes used Ampure beads that were 1-3 years past expiration date. They sort of work, but they seem to become less magnetic and and are therefore more difficult to work with.
Forum: Illumina/Solexa 10-13-2018, 08:22 AM
Replies: 2
Views: 914
Posted By JBKri
Share run between NEBNext smallRNA and 16s amplicons in v3-600 MiSeq run?

Hi all,
for testing purposes we are thinking to put a small amount (~2-3 %) of a smallRNA library (NEBNext) in a run that will otherwise consists only of 16s amplicons (~490 bp, Nextera-type 8 bp...
Forum: Oxford Nanopore 09-26-2018, 02:59 AM
Replies: 0
Views: 1,797
Posted By JBKri
best approach in dealing with low input DNA

Hi everybody,
we already posted this in the Nanopore forums, but we wanted to have a better chance of receiving answers so we post it again here.

We are planning in our lab to perform an...
Forum: Sample Prep / Library Generation 07-04-2018, 01:00 AM
Replies: 5
Views: 1,629
Posted By JBKri
Would anyone happen to have a pdf of this: ...

Would anyone happen to have a pdf of this:
GelStar nucleic acid gel stain: high sensitivity detection in gels.
White HW1, Vartak NB, Burland TG, Curtis FP, Kusukawa N. Biotechniques. 1999...
Forum: Illumina/Solexa 06-25-2018, 02:04 AM
Replies: 3
Views: 1,249
Posted By JBKri
On further investigation I see that the pattern...

On further investigation I see that the pattern varies depending on the number of cycles in the sequencing kit, and also some variation among runs. It seems 2*150 cycles causes little or no sudden...
Forum: Illumina/Solexa 06-19-2018, 09:32 AM
Replies: 3
Views: 1,249
Posted By JBKri
Base composition vs cycle, MiSeq vs HiSeq X

I have noticed that in our MiSeq runs we seems to always have an abrupt change in base composition in the last one or two cycles. The proportion of A goes to zero. Also I noticed we have a cyclic...
Forum: Sample Prep / Library Generation 06-15-2018, 07:20 AM
Replies: 5
Views: 1,629
Posted By JBKri
Hi Chiara, it seems SybrGold is sensitive down...

Hi Chiara,
it seems SybrGold is sensitive down to 25 pg of DNA, see...
Forum: Sample Prep / Library Generation 06-14-2018, 03:46 AM
Replies: 5
Views: 2,557
Posted By JBKri
What type of library is this? How did you measure...

What type of library is this? How did you measure the concentration? For many types of libraries it is best to measure the concentration by qPCR. The resulting concentrations are expressed in pM or...
Forum: Illumina/Solexa 03-01-2018, 03:11 AM
Replies: 7
Views: 2,141
Posted By JBKri
I am facing a similar choice. I have no previous...

I am facing a similar choice. I have no previous experience with MDA, but my thought is to use the Nextera XT kit and thus avoid the MDA. I have the impression that biases from MDA are far worse than...
Forum: Sample Prep / Library Generation 05-30-2017, 04:50 AM
Replies: 12
Views: 6,967
Posted By JBKri
Yesterday I experienced a similar problem, but...

Yesterday I experienced a similar problem, but the clumping (https://goo.gl/photos/rjZuhupF92WLqaeG9) happened in the first step, at the mixing of the bead solution and the DNA. This was HMW genomic...
Forum: Sample Prep / Library Generation 11-11-2016, 07:58 AM
Replies: 14
Views: 10,242
Posted By JBKri
Sheared DNA is end-repaired before size selection...

Sheared DNA is end-repaired before size selection (at least in the TruSeq protocols).
Jon
Forum: Illumina/Solexa 11-05-2016, 06:42 AM
Replies: 5
Views: 1,999
Posted By JBKri
Great, that's just what I was hoping for!

Great, that's just what I was hoping for!
Forum: Illumina/Solexa 11-04-2016, 06:10 AM
Replies: 5
Views: 1,999
Posted By JBKri
Thanks. Does this mean we should do a more...

Thanks. Does this mean we should do a more stringent size selection than the standard TruSeq two-sided magnetic bead size selection? Do you have any links to protocols for this? The Illumina Custom...
Forum: Illumina/Solexa 11-04-2016, 05:26 AM
Replies: 5
Views: 1,999
Posted By JBKri
HiSeq X different insert size?

I am about to prepare TruSeq PCR-free libraries for HiSeq X sequencing.
From:
http://support.illumina.com/sequencing/sequencing_instruments/hiseq-x/questions.html

"The supported insert size on...
Forum: Oxford Nanopore 10-11-2016, 01:02 AM
Replies: 1
Views: 2,037
Posted By JBKri
MinION wash kit, how many washes?

As the title says, how many washes can be performed with one MinION wash kit?
Forum: Illumina/Solexa 08-30-2016, 08:21 AM
Replies: 17
Views: 2,631
Posted By JBKri
One way is to modify the sample sheet to use a...

One way is to modify the sample sheet to use a subset of the length of the indexes (at the cost of possibly increased false assignments). Typically the 8th base of Index1 and the 1st base of index2...
Forum: Oxford Nanopore 07-29-2016, 04:48 AM
Replies: 32
Views: 20,528
Posted By JBKri
Well, at 200x magnification, the liquid loooks...

Well, at 200x magnification, the liquid loooks clean and clear, and the beads look good...
Jon
Forum: Oxford Nanopore 07-26-2016, 08:33 AM
Replies: 32
Views: 20,528
Posted By JBKri
Thanks, this is very useful information. We...

Thanks, this is very useful information.

We do have a bottle of Dynal M270 streptavidin beads, but it expired in 2012. I think the bottle has only been opened once, and it has always been stored...
Forum: Oxford Nanopore 07-22-2016, 06:51 AM
Replies: 32
Views: 20,528
Posted By JBKri
Is it correct that the g-TUBES are optional? ...

Is it correct that the g-TUBES are optional?
Is the Hula mixer really necessary; can't we just mix by hand?
Could you suggest an alternative to the MyOne C1 Streptavidin beads? The smallest bottle...
Forum: Oxford Nanopore 07-19-2016, 02:10 AM
Replies: 32
Views: 20,528
Posted By JBKri
MinION protocols?

Is there a site where I can find detailed protocols for library prep for MinION? A few days ago I signed up for the community, but I still don't have access to the community website. I would like to...
Forum: Illumina/Solexa 07-19-2016, 01:58 AM
Replies: 3
Views: 2,198
Posted By JBKri
I suppose this is the two-step PCR approach? If...

I suppose this is the two-step PCR approach? If so you could also add a short secondary index between the specific primer and the tail, to make it "double-dual" indexing. With two pairs of tailed,...
Forum: Illumina/Solexa 06-09-2016, 02:07 AM
Replies: 5
Views: 2,038
Posted By JBKri
Definitely start with the end repair; it adds a...

Definitely start with the end repair; it adds a phosphate group that probably is necessary!

We usually add about 100 ng of PCR product, and we get around 50% of these fragments with adapters...
Forum: Sample Prep / Library Generation 06-08-2016, 12:14 AM
Replies: 7
Views: 1,948
Posted By JBKri
We are also considering omitting the cleanup. ...

We are also considering omitting the cleanup.
Could you explain a little, what do you mean you are getting 50 %? Are the other 50 % dimers, byproducts etc?
Forum: Sample Prep / Library Generation 06-06-2016, 06:37 AM
Replies: 7
Views: 1,948
Posted By JBKri
What do you mean by reproducibility? The...

What do you mean by reproducibility? The non-template overhang would be the same length if we do the two steps separately, right? I am worried about possible biases, chimeras, long primer-adapter...
Forum: Sample Prep / Library Generation 06-06-2016, 03:43 AM
Replies: 7
Views: 1,948
Posted By JBKri
Two-step PCR in single step?

We would like to try to do the Illumina two-step PCR (1st PCR with universal tail, 2nd PCR adds indexed adapters) in a single PCR reaction, to lessen chances of contamination. According to the old...
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