[haheller]

New to this so my knowledge-base is still quite limited so be gentle in reply, please
We will prepare miRNA-Seq library and send it out for sequencing on a GAII. Can anyone confirm that these adapter/Primer sequences first reported by ECO have (can) been used successfully on the GAII (and presumably GAIIx)?

Small RNA oligonucleotide sequences
RT Primer
5' CAAGCAGAAGACGGCATACGA

5' RNA Adapter
5' GUUCAGAGUUCUACAGUCCGACGAUC

3' RNA Adapter
5' P-UCGUAUGCCGUCUUCUGCUUGUidT

Small RNA PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Small RNA PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Small RNA Sequencing Primer
5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC

Was (is) there a GAI instrument? Does it require different Adapter/Primer sequences? Can't find any evidence of a GAI version on Illumina website.
Thanks in advance

[der_eiskern]

tgdeering, phosphorothioate fixed my problems. before i could barely see a band after amplification but with phosphorothioate primers i got maximal amplification with iProof.

[der_eiskern]

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.

[tgdeering]

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?

[der_eiskern]

thanks greigite!

[greigite]

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).

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.

[der_eiskern]

Genomic PCR Oligo Mix

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.

Sequences for Solexa Library Preparations:

Genomic DNA oligonucleotide sequences

Adapters 1
5' P-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT

PCR Primers 1
5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT

Genomic DNA Sequencing Primer
5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT


DpnII gene expression oligonucleotide sequences

Gex Adapter 1
5' P-GATCGTCGGACTGTAGAACTCTGAAC
5’ ACAGGTTCAGAGTTCTACAGTCCGAC

Gex Adapter 2
5' CAAGCAGAAGACGGCATACGANN
5' P-TCGTATGCCGTCTTCTGCTTG

Gex PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Gex PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Gex Sequencing Primer
5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC

NlaIII gene expression oligonucleotide sequences

Gex Adapter 1
5' P-TCGGACTGTAGAACTCTGAAC
5' ACAGGTTCAGAGTTCTACAGTCCGACATG

Gex Adapter 2
5' CAAGCAGAAGACGGCATACGANN
5' P-TCGTATGCCGTCTTCTGCTTG

Gex PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Gex PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Gex Sequencing Primer
5' CCGACAGGTTCAGAGTTCTACAGTCCGACATG


Small RNA oligonucleotide sequences

RT Primer
5' CAAGCAGAAGACGGCATACGA

5' RNA Adapter
5' GUUCAGAGUUCUACAGUCCGACGAUC

3' RNA Adapter
5' P-UCGUAUGCCGUCUUCUGCUUGUidT

Small RNA PCR Primer 1
5' CAAGCAGAAGACGGCATACGA

Small RNA PCR Primer 2
5' AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA

Small RNA Sequencing Primer
5' CGACAGGTTCAGAGTTCTACAGTCCGACGATC

**disclaimer blah blah blah. We take no responsibility if you blow a flow cell using these sequences!

[kmcarr]

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)

[hilahg]

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

[frozenlyse]

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)

5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
                      |||||||||||||
3'-GAGCCGTAAGGACGACTTGGCGAGAAGGCTAG-PO4-3'

or are they two sets of fully complementary primers, so i should order 4 oligos? this is what it seems to illustrate in post #7

5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
   ||||||||||||||||||||||||||||||||
3'-TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAG-PO4-5'
and
5'-PO4-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3'
       ||||||||||||||||||||||||||||||||
   3'-TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTC-5'

Cheers!

[jrelmore]

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

[jrelmore]

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?

[kwatkins]

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

[wangdaowen]

thank,
Has anyone got the multiplex oligo sequences that Illumina use in their Multiplex (12-plex) kits? (And their concentrations if they're known.)

[wangdaowen]

thank

yes it help,thank