Guys, I know nothing about bioinfo, I need guidance with one of my work. I m more of a wet lab person.
I have to work out primers for certain domains of some proteins related to cancer. How do we go about this.
The only information given to me is the name of the domain of the protein (example BRCA2 DNA binding domain). How do we find the dna sequence position of this domain in the gene and how do we design primer for Sanger sequencing from it?
please help, the people in my institution are very procrastinating in nature and are not helping me to learn. Please guide me and I will learn from it.
thank you in advance kind souls. ❤
What happened exactly that led to the low quality of the chromatogram signal? Thanks in advance
My question revolves around the fluorescent protein attachment. Does it happen post elongation/termination with the ddNTP, or are they bound prior to DNA polymerase recruitment.
If they are bound at the start, how is it able to fit such a selective enzyme as DNA pol??
Get to know Eurofins Genomics. We are introducing Andreas Hinkel, business unit manager of the Sanger sequencing lab. Read here about his work and responsibilities in the high-throughput Sanger sequencing lab: https://the-dna-universe.com/2021/09/01/meet-the-sanger-sequencing-lab-expert-andreas-hinkel/
https://preview.redd.it/nbp9795ftvk71.jpg?width=900&format=pjpg&auto=webp&s=1cdb2e4817883a9c8dc59be51307dfb6ca300cfc
Title says it all. I cannot for the life of me understand it! Any help would be appreciated.
Wanted to let this sub know I have received an update for my previous post if anyone was curious
My genomics center used to have a native viewing software for chromatograms. Anyone have suggestions for viewing Sanger sequencing data? Preferably something free/cheap?
Hello, I am doing a 96-well arrayed cloning. I have done Gibson, transformations and minipreps in a 96-well format, then sent out the plate for sequencing. Normally, I sequence 3-4 clones and check if the sequencing looks good manually with Benchling cause it has nice visualization tool that highlights mismatches but also displays sequence quality. Now I have to check the sequences of 96 individual clones. Obviously I can write a simple script that would do alignments and report the alignment quality. However, I am guessing there should be already tools available that work with Sanger clonal sequences and provide nice visualization for many alignments at once. Any recommendations for specific tools I should use? Thank you!
Hello,
Sanger sequencing seems to use the same method as PCR (denaturation, annealing, elongation...), but PCR was discovered in 1986, sanger method created in 1976 : how is it possible ?
Thank you in advance !
Hi All, I'm getting a bit frustrated with Eurofins and would like to try another company. What is everyone out there using, what do you pay per reaction? I'm in the Northeastern US. Thanks!
I recently used a site directed mutagenesis protocol to create several degenerate codons (NDT) in an 800bp sequence. I have received my sequencing back and while I have no traces of wild type, the peak heights in the electropherogram do not always correspond to the expected ratio of nucleobases, e.g. for an 'N' region I would expect ATCG to be 1:1:1:1 however they look like maybe 1:2:3:1 (just an example).
I am wondering if this is a sanger sequencing thing (how much should I trust relative heights in the electropherogram) or if my construct actually has an uneven distribution of bases in these degenerate regions - any advice ?
I understand what Sanger Sequencing is and what it does but not the big picture of how it works..
I understand that the ddNTPs will cause termination at whatever specific nucleotide it is, resulting in a large amount of fragments of different lengths.
I don't understand how obtaining these multiple different fragments tell us the sequence of the DNA through electrophoresis. I know that smaller fragments travel farther/larger fragments won't travel farther. I get how you read electrophoresis (top to bottom) to determine the sequence if it was given... I just don't understand the concept of how the different fragments resulting in bands for each ddNTP read top to bottom GIVES us the sequence.
Anyone follow? sorry, heard to explain.
https://imgur.com/a/BHL7TU7
As I understand it, the Sanger sequencing results give you the COMPLEMENTARY strand of DNA to your sample. In this case, shouldn't the answer be C? Answer choice C gives us
5' CGTAA 3' which is the complementary sequence to the one in the question stem, which would be accurate for Sanger sequencing. Am I wrong here, or is the question-maker wrong?
Hello! I know that we can't really distinguish between different alleles, but considering a single chromosome, how can we actually differentiate between the two strands from a single chromosome?
Assuming we are sequencing a new genome and have no ability to use primers that would bind one strand or the other.
If we fragment the DNA, do sanger/capillary sequencing which will generate dsDNA fragments of all possible lengths, how could we possibly distinguish between the two strands? We would just get a double signal at each size on the gel/coming through the capillary.
Thanks for any help in advance, this has been killing me...
Hi all,
Why does the chromatogram resulting from Sanger sequencing decrease in signal over the length of the sequence? Also, why is the peak resolution poor at the beginnings and ends of the sequence?
I’m sequencing zebrafish using the following string of protocols:
Extract tissue —> —> Isolate DNA and amplify via PCR —>Purify via Exo-CIP —>Send off for sequencing
I’ve had inconsistent signal to noise ratios coming back for quality. What can be done to improve this? Right now the only thing I can think of is being more careful with contamination
I'm trying to troubleshoot some sanger sequencing results. My chromatograms show gaps scattered within a gene I'm trying to sequence. The chromatogram is correct upstream and downstream of these gaps. I'm thinking the polymerase is slipping off, or could the growing strand be dissociating and then re-annealing?
Thanks for any input.
Does Sanger sequencing give the complementary strand or the template strand?
I've seen conflicting info between UWorld and other resources.
Get to know Eurofins Genomics. We are introducing Andreas Hinkel, business unit manager of the Sanger sequencing lab. Read here about his work and responsibilities in the high-throughput Sanger sequencing lab: https://the-dna-universe.com/2021/09/01/meet-the-sanger-sequencing-lab-expert-andreas-hinkel/
https://preview.redd.it/0lz9clzftvk71.jpg?width=900&format=pjpg&auto=webp&s=ed268715e749eda04c782fd66057b74ec373d499
