Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • I found this page, maybe this is what you are looking for? I hope this helps:



    Best,

    C

    Comment


    • PE primers

      Hi,

      I am looking for PE primer 1.0 and primer 2.0 sequences that people have good experience with. There is too much confusion whether these primers need to be modified at 3' (LCNA or phosphothioate).

      Can anyone share exact modifications for primers and their sequences? I already have lot of adapters that come with PE oligo kit but ran out of primers.

      Thanks
      Shob

      Comment


      • thanks a lot

        Comment


        • Hi, what is the range of DNA reduced representation libraries sequence length, its 200-300bp, the older one, what is the newer length range?.

          Comment


          • Originally posted by dandestroy View Post
            Sorry for the small font, but that's the only way I could make it fit


            Paired-end DNA

            PE Adapter1:
            5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
            3' -------------------- -----TGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGp (-) -------------------- -------------------- -------------------- - 5'
            PE Adapter2:
            5' -------------------- -------------------- ------------------ (-) pGATCGGAAGAGCGGTTCAG CAGGAATGCCGAG------- -------------------- - 3'
            3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTC------- -------------------- - 5'
            PE PCR Primer1:
            5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
            3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5'
            PE PCR Primer2:
            5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3'
            3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5'
            Result Library:
            5' AATGATACGGCGACCACCGA GATCTACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (N) AGATCGGAAGAGCGGTTCAG CAGGAATGCCGAGACCGATC TCGTATGCCGTCTTCTGCTT G 3'
            3' TTACTATGCCGCTGGTGGCT CTAGATGTGAGAAAGGGATG TGCTGCGAGAAGGCTAGA (N) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGCTAG AGCATACGGCAGAAGACGAA C 5'
            PE DNA Sequencing Primer1
            5' -------------------- -----ACACTCTTTCCCTAC ACGACGCTCTTCCGATCT (-) -------------------- -------------------- -------------------- - 3'
            3' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 5'
            PE DNA Sequencing Primer2
            5' -------------------- -------------------- ------------------ (-) -------------------- -------------------- -------------------- - 3'
            3' -------------------- -------------------- ------------------ (-) TCTAGCCTTCTCGCCAAGTC GTCCTTACGGCTCTGGC--- -------------------- - 5'

            Hi all,

            Can anyone point me to a reference for this sequence? following the Illumina TruSeq kit, the final PE TruSeq product doesn't look like this for me. Especially for the top read, after the Ns, where the 3' adapter is, seems to be different.

            Really appreciate any feedback!

            Comment


            • Originally posted by tldgID View Post
              Hi all,

              Can anyone point me to a reference for this sequence? following the Illumina TruSeq kit, the final PE TruSeq product doesn't look like this for me. Especially for the top read, after the Ns, where the 3' adapter is, seems to be different.

              Really appreciate any feedback!
              The library schematic you are looking at is for legacy Illumina adapters not the the TruSeq versions. The original adapters required a PCR step to add the portion of the library that attaches to the Flowcell. The True Seq adapters are complete and there are some sequence differences to the original non-True Seq versions - check the Illumina customer letter for the correct sequemces.

              Comment


              • Originally posted by protist View Post
                The library schematic you are looking at is for legacy Illumina adapters not the the TruSeq versions. The original adapters required a PCR step to add the portion of the library that attaches to the Flowcell. The True Seq adapters are complete and there are some sequence differences to the original non-True Seq versions - check the Illumina customer letter for the correct sequemces.
                Thanks protist! not sure what 'legacy Illumina adapters' are thought! and I couldn't find any information about them on Illumina website or customer letter. But anyways, thanks.

                Comment


                • Originally posted by tldgID View Post
                  Thanks protist! not sure what 'legacy Illumina adapters' are thought! and I couldn't find any information about them on Illumina website or customer letter. But anyways, thanks.
                  The original adapters (what I called legacy) released by Illumina are not exactly the same sequence as the currently in use TruSeq adapters - e.g. old adapters did not have indexes TruSeq ones do. The sequences for the individual TruSeq adapters can be found in the Illumina customer letter (see attached) which can also be downloaded from the Illumina website. I have also attached an alignment I had illustrating the differences between the original adapters and the currently in use TruSeq adapters.
                  Attached Files

                  Comment


                  • This is a great comparison protist! thank you!

                    Comment


                    • Hi,
                      I've been trying to sequence the V3-V4 region of the 16S gene was hoping touse my forward and reverse primers as my sequencing primers and the reverse compliment of the reverse primer as my index primer. The problem I just found during a real time PCR to determine sequencing primer efficiency is that the primers will not bind their targets efficiently at the required temperature (65oC).
                      We can easily increase the length of the sequencing primers going back into the Illumina adapters, but I am afraid that when I increase the length of the index primer I will end up out of the conservative region. I've tried including several degeneracies, but the lowest Tm is never higher than 65oC.
                      the reverse primer I choose is the S-D-Bact-0785-a-A-21: 5′-GACTACHVGGGTATCTAATCC-3

                      I appreciate any ideas or comments.

                      Marcio.

                      Comment


                      • Hi
                        I m new and happy to be part of seqanswers
                        I hope to learne and to be also usful
                        mchikri

                        Comment


                        • Insert length?

                          Can anyone plz tell mE what is the insert length (bp) (assuming adaptor length is 60bp on each end) for 350bp DNA fragment subjected to paired end sequencing.
                          THANKS!

                          Comment


                          • Originally posted by bssharma View Post
                            Can anyone plz tell mE what is the insert length (bp) (assuming adaptor length is 60bp on each end) for 350bp DNA fragment subjected to paired end sequencing.
                            THANKS!
                            230 bp? (350 - (2 x 60))
                            Not entirely clear what you are asking, though. I am presuming by "350 bp DNA fragment" you mean, 350 bp amplicon.

                            --
                            Phillip
                            Last edited by pmiguel; 11-07-2013, 06:55 AM.

                            Comment


                            • Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
                              Thank you so much

                              Comment


                              • Originally posted by pmiguel View Post
                                230 bp? (350 - (2 x 60))
                                Not entirely clear what you are asking, though. I am presuming by "350 bp DNA fragment" you mean, 350 bp amplicon.
                                Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
                                Thank you so much

                                Comment

                                Latest Articles

                                Collapse

                                • seqadmin
                                  Choosing Between NGS and qPCR
                                  by seqadmin



                                  Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...
                                  10-18-2024, 07:11 AM
                                • seqadmin
                                  Non-Coding RNA Research and Technologies
                                  by seqadmin




                                  Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.

                                  Nobel Prize for MicroRNA Discovery
                                  This week,...
                                  10-07-2024, 08:07 AM

                                ad_right_rmr

                                Collapse

                                News

                                Collapse

                                Topics Statistics Last Post
                                Started by seqadmin, Yesterday, 05:31 AM
                                0 responses
                                10 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 10-24-2024, 06:58 AM
                                0 responses
                                20 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 10-23-2024, 08:43 AM
                                0 responses
                                48 views
                                0 likes
                                Last Post seqadmin  
                                Started by seqadmin, 10-17-2024, 07:29 AM
                                0 responses
                                58 views
                                0 likes
                                Last Post seqadmin  
                                Working...
                                X