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Forum: Illumina/Solexa 07-19-2012, 11:28 PM
Replies: 1
Views: 2,308
Posted By Akira
Validation of Custom Sequencing Primer

Hi.

I would like to use my own sequencing primer. I design it with properties (Tm
and GC content and length etc) close to standard Illumina Sequencing Primer.

To check its performance I set...
Forum: Illumina/Solexa 07-04-2012, 12:51 AM
Replies: 7
Views: 3,244
Posted By Akira
Hi. New to NGS here. Can I ask, what is the...

Hi. New to NGS here.

Can I ask, what is the minimum concentration or minimum amount for PCR-enriched library? What I was told is at least 10nM for subsequent works. Not sure the volume needed...
Forum: Sample Prep / Library Generation 05-23-2012, 07:27 PM
Replies: 18
Views: 24,550
Posted By Akira
Hi. I have my own customized oligos and I would...

Hi. I have my own customized oligos and I would like to anneal them to make adapters. I've seen many protocols (including the Meyer's one) and I am confused whether to use Annealing Buffer with EDTA...
Forum: Illumina/Solexa 04-18-2012, 05:57 AM
Replies: 9
Views: 8,777
Posted By Akira
I see. I am now thinking whether to choose oligo...

I see. I am now thinking whether to choose oligo synthesized by IDT or Sigma. Any idea which is better? (*wondering if I could ask this question)
Forum: Illumina/Solexa 04-18-2012, 05:09 AM
Replies: 9
Views: 8,777
Posted By Akira
Thanks for the reply. How about custom oligos for...

Thanks for the reply. How about custom oligos for adapters? Desalted is sufficient enough?
Forum: Illumina/Solexa 04-17-2012, 07:57 PM
Replies: 9
Views: 8,777
Posted By Akira
Desalting is really fine for both custom adapters...

Desalting is really fine for both custom adapters and custom primers? HPLC is quite expensive. :(
Forum: Sample Prep / Library Generation 04-12-2012, 07:58 PM
Replies: 7
Views: 18,278
Posted By Akira
How about paired end and multiplex? Do they have...

How about paired end and multiplex? Do they have same read 2 sequencing primer? Because I can't find the similarity (not sure if the sequence I got for both adapters are the same).

Paired end...
Forum: Illumina/Solexa 04-02-2012, 06:18 PM
Replies: 145
Views: 299,982
Posted By Akira
During PCR amplification, the primer2.0 will...

During PCR amplification, the primer2.0 will first bind to P7 region, synthesize the 1st PCR product, then only primer1.0 will be able to bind to its complement at P5 region, am I right?

What if I...
Forum: Illumina/Solexa 04-02-2012, 08:49 AM
Replies: 145
Views: 299,982
Posted By Akira
Many thanks for the reply. A random...

Many thanks for the reply.

A random thought: Is it possible to make a Y-adapter, using the reverse complement sequence of the fork? Instead of 5' end of the adapter start with P5 region, change...
Forum: Illumina/Solexa 03-29-2012, 01:19 AM
Replies: 145
Views: 299,982
Posted By Akira
Hi. Is it the old PE adapters supplied in Paired...

Hi. Is it the old PE adapters supplied in Paired End Sample Prep Kit contains a mixture of 2 types of double stranded adapters?

While the new TruSeq DNA sample prep kit only comes with one...
Forum: Illumina/Solexa 03-09-2012, 06:54 AM
Replies: 3
Views: 3,157
Posted By Akira
Many thanks for the info! How about the TruSeq...

Many thanks for the info! How about the TruSeq PCR Master Mix? Try that with own custom made primer before? I have that with me now, and it takes time if I were to order from NEB or KAPA, so I am...
Forum: Illumina/Solexa 03-08-2012, 10:58 PM
Replies: 3
Views: 3,157
Posted By Akira
PCR Enrichment - Replace TruSeq PPC and PMM?!

Hi all, NGS newbie here. I know this has been discussed in other threads, but I don't really understand. I have already had the sample until ligation part (using TruSeq DNA Sample Prep Kit), I would...
Forum: Sample Prep / Library Generation 01-12-2012, 06:05 AM
Replies: 8
Views: 5,813
Posted By Akira
Thanks HESmith. In this case, I can actually...

Thanks HESmith. In this case, I can actually revert back the concept by starting to sequence from the other side of the fragments, which is the genomic DNA as read 1, then the transposon end with low...
Forum: Sample Prep / Library Generation 01-12-2012, 05:56 AM
Replies: 8
Views: 5,813
Posted By Akira
Many thanks for the reply. Cluster calling...

Many thanks for the reply. Cluster calling happens in both read 1 and read 2 for paired end sequencing or only read 1? Most of the clusters will be filtered out if the clusters calling was not good...
Forum: Sample Prep / Library Generation 01-12-2012, 05:46 AM
Replies: 8
Views: 5,813
Posted By Akira
Many thanks to the reply. :) If paired end,...

Many thanks to the reply. :)

If paired end, will the cluster calling be repeated for read 2? Or once the first four cycles of read 1 have coordinated the clusters position, it doesn't matter if...
Forum: Sample Prep / Library Generation 01-12-2012, 02:39 AM
Replies: 8
Views: 5,813
Posted By Akira
Question Sequence transposon flanking region

I am new to NGS first of all. I would like to sequence transposon flanking region, thus during PCR amplification step, instead of using primers supplied by Illumina TruSeq, I will use own customized...
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