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Thread | Thread Starter | Forum | Replies | Last Post |
trim adapter from Illumina Genome Analyzer IIe miRNA reads | NicoBxl | Bioinformatics | 5 | 01-02-2014 05:31 AM |
Illumina Adapter and Primer preparation | S.Iyengar | Sample Prep / Library Generation | 7 | 11-04-2013 07:08 AM |
primer/adapter sequences | nikiwilson | Sample Prep / Library Generation | 2 | 06-21-2011 01:36 PM |
what's the different between Illumina Genome Analyzer | biocc | Illumina/Solexa | 3 | 06-03-2010 11:28 PM |
A clarification for Illumina/Solexa Genome Analyzer Primer/Adapter Sequences | kaichen | Illumina/Solexa | 1 | 08-06-2009 05:57 PM |
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#21 |
Junior Member
Location: asia Join Date: Mar 2009
Posts: 4
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Hi all,
I have a question: I've prepared a cDNA library for solexa (double stranded) with 5' end after enzymatic cleavage by MMEI (see below). I would like to perform a single read from 5' (the left side with the MME sticky end). GEX2 adapter fits this site. My question is which adapter do I need for the 3' side to perform a single read of the 5' side (left side)? 5'_________? 3' adapter Gex 2 3'___________ (MMEI side) which adapter on this side? Can anyone answer this??? |
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#22 |
Junior Member
Location: asia Join Date: Mar 2009
Posts: 4
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the figure came out wrong... I hope you can still understand my question above....
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#24 |
Junior Member
Location: asia Join Date: Mar 2009
Posts: 4
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yes it helps, thank you
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#25 |
Senior Member
Location: Cambridge, MA Join Date: Mar 2009
Posts: 141
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Perhaps the sequences are shown in Figure 2 and table S3 of this paper by Craig et al (http://www.nature.com/nmeth/journal/...th.1251.html)? I notice one of the authors has an Illumina affiliation and the paper describes 6 bp error corrected tags. Of course, they don't tell you outright which ones are the best but my guess is the 13 tags showing less than 2-fold difference in index frequencies between runs (Fig 2).
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#26 |
Senior Member
Location: Cambridge, MA Join Date: Mar 2009
Posts: 141
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Hi all
I put together a comparison of SR and PE sequences to get a quick look at regions of sequence similarity. Thought it might be useful for others (let me know if you spot errors). As far as I can tell, the SR and PE read 1 sequencing primers are identical, as are the SR and PE adapters with the T overhang. Seems to me that given this, it should be fine to run a PE library on an SR flowcell using SR reagents. Comments? All the sequences are from the Bentley et al. nature paper supplementary info last year. |
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#27 |
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Location: china Join Date: May 2009
Posts: 2
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yes it help,thank
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#28 |
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Location: china Join Date: May 2009
Posts: 2
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thank,
Has anyone got the multiplex oligo sequences that Illumina use in their Multiplex (12-plex) kits? (And their concentrations if they're known.) |
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#29 |
Junior Member
Location: Providence, RI Join Date: Jun 2009
Posts: 1
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Hi!
I am trying to add adapters onto a cDNA pool by PCR. Has anyone done this before? I not sure how to design and orient my primers. I am currently looking to use the adapters for "Genomic DNA oligonucleotide sequences" Thanks! Kate |
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#30 |
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Location: Athens, GA Join Date: Jul 2009
Posts: 2
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Does anyone happen to know the concentrations of the PR 5' adapter and the 10x v1.5 sRNA 3' adapter in the small RNA cloning kits?
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#31 |
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Location: Athens, GA Join Date: Jul 2009
Posts: 2
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I know a few people other than myself were looking for the concentrations of the Solexa v1.5 kit (and well all of them in my case) adapters. I spoke with a tech support person from Illumina and they told me the concentrations of the stock solutions before any of the dilutions were as such:
3' sRNA Adapter v1.5 = 5µM SRA 5' Adapter = 5µM Other oligos: SRA RT primer = 100µM Primer GX1/2 = 25µM Hope that helps! -Joshua |
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#32 |
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Location: Australia Join Date: Sep 2008
Posts: 136
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Hi - I am a bit confused about the Illumina Paired end adaptors..
Are they a Y adaptor (illustrated below) where you have 2 primers which are complementary at the end you ligate to your library (ie i should only synthesise 2 oligos) Code:
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' ||||||||||||| 3'-GAGCCGTAAGGACGACTTGGCGAGAAGGCTAG-PO4-3' Code:
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' |||||||||||||||||||||||||||||||| 3'-TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAG-PO4-5' and 5'-PO4-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3' |||||||||||||||||||||||||||||||| 3'-TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTC-5' |
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#33 |
Junior Member
Location: asia Join Date: Mar 2009
Posts: 4
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Hi all,
thank you for all the replies. It was very helpful. I have another question regarding MME cleavage. I use this protocol for library preparation(with MMEI cleavage): http://keck.med.yale.edu/microarrays...04241_RevA.pdf I'm experiencing trouble with the MME cutting procedure as it doesnt! cleave. Has anyone experienced this problem in the library preparation process. I use MME from NEB and tried various concentrations but without improvement... Thanks, Hilah |
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#34 |
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Location: USA, Midwest Join Date: May 2008
Posts: 1,143
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frozenlyse,
You would only synthesize the two oligos as in your first figure. Look at this thread http://seqanswers.com/forums/showthread.php?t=1169 (in particular the PDF attachment) |
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#35 | |
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Location: California Join Date: Jul 2009
Posts: 46
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so it's friday evening, and i was supposed to be amplifying my library before going home tonight however my coworker used all of our amplifying PCR Primers 2.1 and 1.1 for SE sequencing and I can't find an accurately labeled figure of the sequences for these SE PCR primers that Illumina sends out. I'm finding PE sequences everywhere though.
clarification? Thanks. ![]() Quote:
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#36 | |
Senior Member
Location: Cambridge, MA Join Date: Mar 2009
Posts: 141
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You can get the SE and PE PCR primer sequences from the supplementary info of Bentley et al 2009 (http://www.ncbi.nlm.nih.gov/pubmed/18987734). I pulled these and did a comparison of PE and SE primers/adapters (p 9 of attached pdf).
Quote:
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#37 |
Member
Location: California Join Date: Jul 2009
Posts: 46
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thanks greigite!
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#38 |
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Location: Texas Join Date: Mar 2009
Posts: 1
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I am looking into making my own SE primers and noticed in the Bentley et al 2009 paper that they used phosphorothioate modified primers. Does anyone else who uses their own primers do this? Also, what purification for the oligos is needed? Standard desalting, PAGE, etc?
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#39 |
Member
Location: California Join Date: Jul 2009
Posts: 46
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i just got HPLC purified phosphorothioate primers today in the post. I'll try to post how happy i am with them.
i've run into problems after gel purifying the ligated adaptors with inefficient amplification, hopefully this helps... we'll see. |
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#40 |
Member
Location: California Join Date: Jul 2009
Posts: 46
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tgdeering, phosphorothioate fixed my problems. before i could barely see a band after amplification but with phosphorothioate primers i got maximal amplification with iProof.
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