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Forum: Sample Prep / Library Generation 05-25-2010, 01:53 PM
Replies: 5
Views: 3,903
Posted By seqAll
Thanks a million for the detail explanation! Very...

Thanks a million for the detail explanation! Very informative, cover many aspects from lab persons' perspective!

And, very useful to know "the range of optimal cpb would be 0.1-1 cpb, rather than...
Forum: 454 Pyrosequencing 05-13-2010, 12:42 PM
Replies: 1
Views: 1,847
Posted By seqAll
Hi aleferna, Copying from a paper...

Hi aleferna,

Copying from a paper (http://nar.oxfordjournals.org/cgi/content/full/gkq332v1?view=long&pmid=20435675) "Using only one nanogram of fragmented DNA, we prepared ... 53.6 millions Y...
Forum: Sample Prep / Library Generation 05-13-2010, 12:20 PM
Replies: 5
Views: 3,903
Posted By seqAll
Other threads in this forum had mentioned that...

Other threads in this forum had mentioned that the Rapid libraries contain some hairpin, by which PCR amplification is somewhat inhibited. The 4th paper (Taqman MGB qPCR paper) showed AB-library and...
Forum: Sample Prep / Library Generation 05-06-2010, 01:22 PM
Replies: 5
Views: 3,903
Posted By seqAll
which qPCR to use for quantitating lib?

Hi,

We want to do qPCR quantitating lib. So for the different kinds that I am aware of:

SYBR gree qPCR (From micrograms to picograms: quantitative PCR reduces the material demands of...
Forum: Literature Watch 05-06-2010, 12:41 PM
Replies: 3
Views: 1,737
Posted By seqAll
Good idea, thanks!

Good idea, thanks!
Forum: Literature Watch 05-06-2010, 10:24 AM
Replies: 3
Views: 1,737
Posted By seqAll
We have been considering using KAPA qPCR for...

We have been considering using KAPA qPCR for quatitating our lib. Has anyone done some comparisons among different qPCRs? Anyone uses KAPA kit and how much copy-per-bead is optimal?
Forum: 454 Pyrosequencing 03-17-2010, 09:05 AM
Replies: 69
Views: 25,533
Posted By seqAll
Why are the DNA capture beads much more efficient...

Why are the DNA capture beads much more efficient than before? Thanks!
Forum: 454 Pyrosequencing 03-05-2010, 12:35 AM
Replies: 69
Views: 25,533
Posted By seqAll
Hi LMcSeq, Do you have leftover enrichment...

Hi LMcSeq,

Do you have leftover enrichment beads? If so, you can run normal PCR with a few of these beads as template, using the amplification primers, for a few cycles (with the same cylcling...
Forum: The Pipeline 03-03-2010, 01:07 AM
Replies: 10
Views: 7,375
Posted By seqAll
The article cited by ScottC: "At the heart of...

The article cited by ScottC:
"At the heart of the desktop-size sequencer is a 9 by 9 millimeter semiconductor chip, currently consisting of an array of about 1.55 million 3.5-micrometer wells ...
...
Forum: Illumina/Solexa 01-17-2010, 02:33 AM
Replies: 4
Views: 3,140
Posted By seqAll
Thanks shurjo, that was very helpful! Does...

Thanks shurjo, that was very helpful!

Does it mean that, in the Illumina protocol, Klenow exo- adds a single A to ~50% of the blunted ends?
Forum: Sample Prep / Library Generation 01-13-2010, 09:20 PM
Replies: 2
Views: 2,804
Posted By seqAll
Sorry, I was not clear. We normally do...

Sorry, I was not clear.

We normally do AMpure beads selection for small size frags. We used gel cut only once in the very beginning, and never do that again.
Forum: Sample Prep / Library Generation 01-13-2010, 02:05 AM
Replies: 2
Views: 2,804
Posted By seqAll
Removal of large fragments?

Is it necessary to remove large fragments from the library?

The amplification, emPCR or bridge PCR, is able to generate amplicons longer than 1kb. During sequencing, would these long amplicons...
Forum: Sample Prep / Library Generation 12-27-2009, 02:27 AM
Replies: 15
Views: 6,142
Posted By seqAll
Hi Shurjo, I would appreciate if you can...

Hi Shurjo,

I would appreciate if you can guide me to some protocols using hexamer primers, or share your protocol. I am quite new to this field.

Best,
seqAll
Forum: General 12-15-2009, 03:56 PM
Replies: 8
Views: 10,269
Posted By seqAll
Thank you Phillip and kmcarr! I have...

Thank you Phillip and kmcarr!

I have checked its website again, "The Genome Analyzer IIx offers a unique combination of 2 x 100 bp read length and >300 million reads per flow cell..."

That is...
Forum: General 12-15-2009, 02:31 AM
Replies: 8
Views: 10,269
Posted By seqAll
Thank you for the information. It sounds...

Thank you for the information.

It sounds like SOLiD will focus on short read. What is the length of Solexa? Illumina website says 75 bp x 2. Does it mean that it is suitable for my samples (about...
Forum: General 12-14-2009, 06:38 AM
Replies: 8
Views: 10,269
Posted By seqAll
read length of SOLiD and Solexa

Hi there,

What is the current read length of SOLiD and Solexa? And will it be increasing in the near future? I am looking for one able to do 150 bp.

Thanks!
Forum: 454 Pyrosequencing 12-04-2009, 08:59 AM
Replies: 9
Views: 49,948
Posted By seqAll
Thanks Philip, that clears my confusion.

Thanks Philip, that clears my confusion.
Forum: 454 Pyrosequencing 11-24-2009, 11:54 AM
Replies: 9
Views: 49,948
Posted By seqAll
Thanks Jason, One question I don't...

Thanks Jason,

One question I don't understand. Most of the time we have the filtered% around 50% to 70%. If the overwhelming majority of beads (2 M per region) loaded do end up in wells of the...
Forum: 454 Pyrosequencing 11-24-2009, 08:12 AM
Replies: 9
Views: 49,948
Posted By seqAll
I see that the numbers of beads loaded and pulled...

I see that the numbers of beads loaded and pulled off do not adds up to the original total.

Is the variation of the bead counter big or small? Maybe I was too warry about the accuracy of bead...
Forum: 454 Pyrosequencing 11-24-2009, 02:26 AM
Replies: 9
Views: 49,948
Posted By seqAll
Interesting question! Would like to know the...

Interesting question! Would like to know the answer as well.

It seems like the way of beads deposition has a large space to optimise. Too dense beads, say if you can fill all wells with beads,...
Forum: Sample Prep / Library Generation 11-23-2009, 09:32 AM
Replies: 27
Views: 12,055
Posted By seqAll
Just wonder how precise the number 99% is. It was...

Just wonder how precise the number 99% is. It was said >99%. But maybe 98.5%, 99.9%, 99.99, ...? So, that explains maybe 2, 3, 4...(unlikey to be 6 though) orders?

Loss or damage in other steps...
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