*Help Desk chat transcript. Issue can be closed if question was resolved in chat.*
Chris: Hello Yomari, how can I help?visitor_name (visitor_email): Where do I go to look for a promoter sequence?Chris: Well, there's a couple of places. Are you simply looking for sequence upstream of a particular gene or transcript?visitor_name (visitor_email): sequence upstream of asp-5Chris: OK, then the first thing I would suggest is to go to the asp-5 gene pageChris: http://www.wormbase.org/species/c_elegans/gene/WBGene00000218#0-g9-3Chris: You can see that there are two transcripts in the "Transcript" column of the "Sequences" widgetChris: Looking at the gene structure in the "Location" widget, you can see that each has a slightly different starting positionvisitor_name (visitor_email): yup, I see itvisitor_name (visitor_email): okayChris: If you click on the gray double helix icon next to the respective transcript nameChris: you can see the unspliced and spliced sequence for the transcriptChris: below the conceptual translation sequence of the protein, there's a section "view unspliced + UTR + 2000 upstream + 2000 downstream"Chris: If you open that you will see 2000bp of upstream sequence in a tan/beige colorChris: You can copy and paste that sequence; you can choose to include the UTR in your upstream sequence or notvisitor_name (visitor_email): Okay, I have a question. Maybe you can help me with it. I'm a high school student, a senior doing an internship at UMass Medical School. We made primers for gateways cloning, but my mentor has asked me to look for the primers localization. I do not know what he means can you help me?Chris: OK, sure. It would be good to ask your mentor, so that you can be sure, but he probably is asking about where in the sequence of the gene the primers matchChris: If you have primer sequences, we have a tool that can help you find itvisitor_name (visitor_email): He has left to take a class, and I don't know how to find that.. because we took att site sequences and connected it to the promoter sequence of asp-5.visitor_name (visitor_email): So the primer sequence is the att sites sequence. I have that.Chris: OK, well Gateway att site sequences are generic and shouldn't really match anywhere specifically in the C. elegans genomeChris: The att sites are added to the beginning (5' end) of the sequence-specific primers that actually target the sequence you're trying to clonevisitor_name (visitor_email): Yeah, I think he wants like the localization of the beginning base pair of that promoter sequence.Chris: OK, has he defined what he means by promoter? Do you understand what he meant by promoter? Some people mean the sequence upstream of the gene up to the ATG (start codon), others might mean up to but not including the UTRvisitor_name (visitor_email): So he means the sequence between asp-12 and asp-5 until atg uncluding the utrvisitor_name (visitor_email): including I meantChris: OK, goodChris: And you have primer sequences, or just the att sites?visitor_name (visitor_email): att sitesChris: OKvisitor_name (visitor_email): that cant be our primers?Chris: the att sites on their own will not recognize any particular sequence in the C. elegans genomeChris: they are only used as an adapter/tool for the Gateway cloningChris: Can you send me what primers you have?visitor_name (visitor_email): okay, so what do I need? if we are trying to connect it to that upstream sequence of asp-5visitor_name (visitor_email): and okayvisitor_name (visitor_email): Forward Primer: ACAACTTTGTATAGAAAAGTTGChris: well, first you need to determine the promoter sequencevisitor_name (visitor_email): Reverse: AGCCTGCTTTTTTGTACAAACTTGTvisitor_name (visitor_email): Okay, I have the promoter sequenceChris: OK, 2000bp upstream of asp-5 ATG?Chris: 2000bp + UTR I meanvisitor_name (visitor_email): yeah, we used the sequence in between asp-12 and asp-5Chris: So, you already have the sequence? Or you talked about using the sequence between asp-12 and asp-5?visitor_name (visitor_email): Yes I have that sequenceChris: OK, can you send it to me?visitor_name (visitor_email): surevisitor_name (visitor_email): ttttcttcagtgtgtgatctacgtgattatggtcaaaagtccaacttatacatatcgtcatgtacaccttatttcttttttcattttctggacaattttgtgaagtgaaaacccacgtcacagaccgttatctgattgagtataaatgataagaaatcattgtcaactcaaataaaataggaatttgttaacttgtagtacgaattctccttattcttatctatttcatagactgagctgtttcattcgtattaccttttttctgtgttttgtcagcttttcctactatttataatttaatgttaactttcacgaccatttttccaggtChris: OK, thanksvisitor_name (visitor_email): Thank you for helping me.Chris: You're welcomeChris: Is your mentor then asking you how to construct the primers?Chris: you would want to add the forward att site to the beginning of the promoter sequence you just sent meChris: and the reverse att site to the end, but the reverse would need to be in reverse complementvisitor_name (visitor_email): He asked me to construct the primers. Assuming that those sequences go together. I used the att sequence in the beginning of the sequence I sent you for my forward primer and the other att sequence at the end and got the reverse compliment to use it as my reverse primervisitor_name (visitor_email): Yeah I did that.Chris: OK greatvisitor_name (visitor_email): so that is correct/visitor_name (visitor_email): ?*Chris: Well, if he's asked you to construct the final primers, you would need to choose about 20-25 nucleotides of target/promoter sequence and use that as the target primer sequence at the end of the att sitevisitor_name (visitor_email): So it would be another primer sequence?visitor_name (visitor_email): that connects to the other?Chris: It would be the forward att sequence followed by the beginning (20-25nt) of your promoter sequencevisitor_name (visitor_email): ohhh okay I got you nowChris: and the reverse att site followed by the reverse complement of the last 20-25 nt of the promoter sequencevisitor_name (visitor_email): Also, sorry I'm being so dense. Now I need the localization of the 5' end of that sequence I sent you.visitor_name (visitor_email): How can I find that?Chris: it's the beginning of the sequence; DNA sequences are always read left-to-right 5' to 3'Chris: so the beginning is 5'Chris: and the end is 3'visitor_name (visitor_email): yes, I need to find the localization of that sequence in the c. elegansvisitor_name (visitor_email): of the 5' and 3'Chris: you mean where in the genome?visitor_name (visitor_email): yesChris: OK, well you can use the BLAST toolChris: http://www.wormbase.org/tools/blast_blat/runChris: SorryChris: http://www.wormbase.org/tools/blast_blatvisitor_name (visitor_email): I just enter the sequence, that I sent you?Chris: paste in your promoter sequence into the boxChris: yesChris: choose "Nucleotide" as the "Query Type", and click on the "BLAT" radio button and click on the "Submit" buttonvisitor_name (visitor_email): Okay..Chris: This will help you see how your sequence aligns to the genome with respect to asp-5 and asp-12visitor_name (visitor_email): Okay, and hwo do I know the localization, for example what chromosome and stuffChris: When you get to the BLAST/BLAT results page, look for the best match where the "Score (bits)" is highest and the "E value" is lowestChris: The first hit I get says "V" and has links to "Alignment" and "Genome View (GBrowse) or (JBrowse)"Chris: that means the top hit is on chromosome V (5)visitor_name (visitor_email): this is so confusing...visitor_name (visitor_email): I'm sorryChris: It's OKChris: What do you see on the BLAST results page?visitor_name (visitor_email): its just that he wants the exact location of that first basepair and last.... you know what I mean? like the chromosomes and then the number that comes aftervisitor_name: uploaded file: https://olark-file-uploads.s3.us-west-1.amazonaws.com/processed/0792b719-2403-4693-951a-6325c8300c33.pngChris: OK, click on the "(GBrowse)"visitor_name (visitor_email): okayvisitor_name: uploaded file: https://olark-file-uploads.s3.us-west-1.amazonaws.com/processed/33b4bad5-cb8d-4422-8607-f0ffd3707cb2.pngChris: I think the answer you're looking for is V:8,272,060..8,272,389Chris: I get this by clicking and dragging the cursor over the "Hit" regionvisitor_name (visitor_email): I was just about to send you thatlmlChris: and zooming inChris: You can zoom way in to the beginning and end of the Hit sequence and see what location number matchesChris: I think the actual beginning is V:8,272,058Chris: and the end is V:8,272,386Chris: You can zoom in specifically by clicking and dragging along the number line at the top of the genome browserChris: look for the specific bp number that corresponds to the beginning and end of you sequence matchvisitor_name (visitor_email): thanksvisitor_name (visitor_email): so muchChris: OK, you're welcomeChris: does that get you what you need?visitor_name (visitor_email): It sure does thatnkyouChris: OK, greatChris: Anything else I can help you with?visitor_name (visitor_email): Nope, you have done enough thank you.Chris: OK, you're welcome. Have a nice day!
**Reported by:** yoma********* (rive******************)
**Submitted from:** https://www.wormbase.org/#012-34-5
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