Forum: Illumina/Solexa
12-04-2012, 05:54 AM
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Replies: 0
Views: 213
Ghost sequences
Hi Friends
Custom protocol sequencing gave 90% junk sequences. Rest is matching the human genome. Majority of junk sequences are repeats of adapter/primer, where the sequencing primer should bind....
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Forum: Illumina/Solexa
12-03-2012, 09:48 AM
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Replies: 6
Views: 324
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Forum: Illumina/Solexa
12-03-2012, 05:53 AM
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Replies: 6
Views: 324
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Forum: Illumina/Solexa
12-03-2012, 02:56 AM
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Replies: 6
Views: 324
Thanks for the reply, yaximik!
I called on...
Thanks for the reply, yaximik!
I called on Illumina techsupport on this question. They say there should not be any problem for PCR products with PS bonds to form clusters. Although they did not...
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Forum: Illumina/Solexa
11-30-2012, 11:18 AM
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Replies: 6
Views: 324
PS bonds and polymerase
Hi folks
I am planning to use illumina primers RP1 and RPI1 with phosphorothioate bonds between the last base and the penultimate base at the 3' end. So my eventual PCR library will contain two...
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Forum: Illumina/Solexa
08-02-2012, 06:13 AM
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Replies: 0
Views: 360
low library concentration
Hi Friends
I am using TruSeq whole RNA sequencing kit. I used 50 ng input of earlier purified mRNA and gave a 15 cycle. At the end, the libraries were dissolved in 30 microliter of RSB buffer. Out...
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Forum: Illumina/Solexa
07-31-2012, 09:12 AM
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Replies: 2
Views: 371
Hi there
I am talking about a pGEM-T clone...
Hi there
I am talking about a pGEM-T clone obtained from a Truseq library. When I blast the sequence with Refseq RNA db using BLASTN, it gives no significant hit. However, after BLASTX it had some...
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Forum: Illumina/Solexa
07-31-2012, 08:23 AM
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Replies: 2
Views: 371
Mitochondrial genome in Tru RNAseq
Hi folks,
What percent of a RNAseq library can be mitochondrial genome? I understand that it should not be there after DNase I treatment. Despite, I have done 2 rounds of oligo dT, I am seeing...
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Forum: Illumina/Solexa
07-27-2012, 04:04 AM
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Replies: 9
Views: 3,015
Hi folks,
How is the following multiplexing...
Hi folks,
How is the following multiplexing (Barcodes 2, 3, 4, 5, 18, 19, 12, 13, 8 libraries) in a lane for TruSeq? In case, it is the problem to pool 2, 3 or 4 libraries I may be safe.
B.No....
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Forum: Illumina/Solexa
07-23-2012, 12:40 PM
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Replies: 0
Views: 599
barcode sequencing primers
I used the RP1 and RPI1, 2, ..... primer/adapter sequences from the Illumina Customer letter for a custom library. Essentially, these are given for the TruSeq Small RNA sequencing strategy.
I...
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Forum: Bioinformatics
05-16-2012, 09:33 AM
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Replies: 2
Views: 215
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Forum: Bioinformatics
05-16-2012, 04:22 AM
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Replies: 2
Views: 215
Reads for a gene
Hi Friends
I am interested to know whether an isoform of a protein (at transcript level) is expressed in different human tissues or cells. My plan is to dig the already available deep sequencing...
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Forum: Sample Prep / Library Generation
05-11-2012, 06:40 AM
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Replies: 7
Views: 647
Hi all,
Great to see that you guys are...
Hi all,
Great to see that you guys are keeping this thread alive. As pointed out by Phillip, I am looking for the cut-off of the single stranded DNA molecules on AMPure beads. Comments are always...
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Forum: Sample Prep / Library Generation
05-10-2012, 02:40 AM
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Replies: 7
Views: 647
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Forum: Illumina/Solexa
05-10-2012, 02:20 AM
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Replies: 2
Views: 526
Thanks Dario. We already thought of a similar...
Thanks Dario. We already thought of a similar CAGE technique exactly from RIKEN labs, however due to the number of steps we were kind of reluctant. Anyhow, I would like to get an answer for my query....
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Forum: Illumina/Solexa
05-09-2012, 03:01 PM
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Replies: 2
Views: 526
5' end tags
Hi Friends
We are interested in the 5' ends of the transcripts. I was thinking of setting an RNA adapter at the 5' end (as in directional sequencing), then shearing the RNA with Mg+2 at 94degC for...
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Forum: Sample Prep / Library Generation
05-09-2012, 12:33 PM
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Replies: 7
Views: 647
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Forum: Sample Prep / Library Generation
05-09-2012, 09:54 AM
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Replies: 7
Views: 647
AMPureXP for ssDNA
Hi Friends
Has anyone used AMPureXP for ssDNA purification? I need to get rid of smaller cDNAs (<100 ntd).
Biochembug
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Forum: Illumina/Solexa
03-02-2012, 08:50 AM
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Replies: 3
Views: 1,080
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Forum: Illumina/Solexa
02-27-2012, 07:50 AM
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Replies: 3
Views: 1,080
Enrichment PCR failure!
Hi Friends
Since a long time now I have been fiddling around some Illumina Truseq primers for a custom sequencing protocol. I am using RP1 and RPI1 (and other indexing primer for my amplification...
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Forum: RNA Sequencing
10-09-2011, 02:55 AM
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Replies: 6
Views: 782
Thanks
Hey that is a good information. I did find the paper. Thank you once again.
Bug
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Forum: Illumina/Solexa
09-26-2011, 06:17 PM
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Replies: 0
Views: 739
sequencing homopolymers
I have few technical queries regarding sequencing some repetitive sequences such as homopolymeric stretches of any base exactly near the start of sequencing primer on Illumina sequencing platform.
...
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Forum: Illumina/Solexa
09-22-2011, 04:56 AM
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Replies: 1
Views: 558
PolyA on flowcell
Hi Friends
If I plan to clone RNA near its 3' end and somehow select just the 3' ends. What do you think about clusters when sequencing the A tail. I am planning to sequence from the back-end of...
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Forum: Illumina/Solexa
09-16-2011, 05:30 PM
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Replies: 39
Views: 7,742
Thanks for reply
Hi Friends
Thanks all for posting replies. Some people whom I talked here at my place suggested qPCR can be best. Anyone knows whether some company provides any standard for it? Of course I would...
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Forum: Illumina/Solexa
03-04-2011, 08:06 AM
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Replies: 0
Views: 726
troubleshooting illumina seq
Dear friends
I have my library which is prepared using custom made primers published in a an article. My eventual product (double stranded of course) will look like this:
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