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DNA Sequence Analysis - Science method

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Last years I’ve been using the classics Bioedit, MEGA, Arlequin, Dnasp, Haploviewer, JModelTest... to manipulate and compute statistics or NJ trees from sequence data. I wonder whether there is a good alternative (or a brief selection) of packages in R to do the same thing.
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Thanks both for your answers. I did not know the Bioconductor project, but it seems promising Jochen Wilhelm. I've seen that msa packahe is also implemented in bioconductor.org, so I will definitely take a look!
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I am analyzing a set of DNA sequences and I would like to use DnaSP6 software on a PC using Ubuntu linux.
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Because it was built on Microsoft, you need to use a virtualBox in case of Unix based OS.
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I have been stuck to find lncRNA tools to annotate the file that have DNA sequence in them. I have four GEO accession which are GSE55191, GSE89186, GSE58043, and GSE64631. All four of them have done the GEO2R analysis separately and then combine the GEO2R result using the Venn diagram to gain the overlapping ID. So, I want to annotate the sequence which I can access using Windows.
Or is there any other way to use the DNA sequence for lncRNA research?
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Thank you for the suggestions!
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Hi everyone, I am new to molecular work and I want to know how to use the Unite database for my dna sequences. Is there a tutorial for complete beginners?
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If you simply want to perform a homology search, you will find an option of run analysis on UNITE homepage. Simply paste your sequences into the correct box.
Cheers
Pankaj
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  1. Telomeres are distinctive structures found at the ends of our chromosomes. They consist of the same short DNA sequence repeated over and over again. They protect the ends of our chromosomes by forming a cap, much like the plastic tip on shoelaces. If the telomeres were not there, our chromosomes may end up sticking to other chromosomes. When the telomere becomes too short, the chromosome reaches a ‘critical length’ and can no longer be replicated. This ’critical length’ triggers the cell to die by a process called ((apoptosis)) also known as programmed cell death.
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Read a review article or two on the subject, the exact mechanism has been described in detail.
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1) I need an explanation of the following statement from a protein expression review article
" The only drawback of these vectors is that they typically have only a single restriction site for N-terminal cloning (NcoI). Thus, if you have a NcoI site in the DNA sequence of your “to-be-expressed” protein, then you cannot use these vectors for cloning unless they are genetically modified to incorporate a new restriction site. Because proteins often have multiple NcoI sites, including proteins we are studying in our laboratories, we modified a subset of these vectors to include an NheI site immediately following the NcoI site, allowing these modified vectors to be used to subclone DNA sequences with NcoI sites"
My question is why vectors having just one NcoI cloning site cannot be used to subclone DNA sequences with NcoI sites? and what the difference that the other site (Nhel) would make to fix this issue?
2) when to use a restriction enzyme that creates a blunt or sticky ends? I know the difference between these ends, but I do not understand why to use a restriction enzyme that creates one of these two specific ends?
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We just need enzyme sites (NCOI) at 2 ends of the insert then after digesting, they can be ligated with the vector. And enzyme sites at 2 ends of the insert can be created by adding the sequence of enzyme sites into the 5' ends of your PCR primers which you use for amplifying the inserts. You can click on the links Manuele Martinelli cited in his comment to get more information.
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I'd like to engineer a probiotic to secrete GHRP-6. Because GHRP-6 contains several D-form amino acids, could it be possible to transform bacteria with DNA sequences encoding the 6 same amino acids having the same function? That is, does GHRP-6 composed of all L-form amino acids have the function? What's the purpose of D-form amino acids in GHRP-6?
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This paper describes the structure of the ghrelin receptor and its interactions with the ligand:
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Where can I find Monkeypox sequences collected in the recent scattered outbreak, I am aware that NCBI has some sequences posted. but is there any database specificely concerned with Monkeypox
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We would like to know the best value for money commercial company for DNA sequencing as part of an RNA-seq study.
Thanks you. Joe Duffy
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Perhaps the LatchBio platform might be of use? It does not sequence data but it's a great RNA-seq analysis pipeline.
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I have pdb files of RNA, but need to convert to DNA sequence. How to manually substitute -OH to H using pymol? &
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Assalamualaikum Nornazliya Mohamad
1. u can use Builder option in Pymol > atoms > delete > click at OH. once the OH was deleted, go to atoms > add H
2. Later u can check the overall structure to ensure it is completely converted to DNA form
just my 2cents :)
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In the entire primer sequence there is a nucleotide that does not match its complementary base in the template DNA sequence.
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If the mismatched base is at the 3'end of the primer or 1 base in from the 3' end of the primer then you may have problems getting the pcr to work. If the mismatch is further into the primer, near the middle or anywhere in the 5'half of the primer then just lower the annealing temperature of that primer from the temperature expected of the whole primer and the pcr will work fine. If you have a gradient pcr machine then running a gradient of annealing temperatures is ideal. Generally if you lower the annealing temperature enough then all single base changes should amplify.
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Through Arlequin, I'm hoping to learn how to study population genetics (e.g., Fst values, Hardy-Weinberg analyses) using DNA sequences. Perhaps you could give screenshots of the files you have or a step-by-step guide on how to perform it. All I know is how to use Arlequin to analyze microsatellite data.
Your response will be greatly appreciated!
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One easy way is to load the entire FASTA (or Nexus) file in DnaSP software and define sequence sets. Defininf sequence sets simply means grouping samples (in this case sequences) into the defined populations. To do this in DnaSP, go to Data<Define sequence sets, then assign sequences to their respective populations. After this, you need to save the defined sets as *.hap and *.arp files. Then load the *.arp file into Arlequin, select the parameters you are interested in, and run. The results (look for file named _main.htm) are written in the folder with the *.arp file. Good luck!
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Hi, I had recently two non-standard DNA sequencing outputs. I'd be curious if anybody could advice what could possibly go wrong, so that I can avoid such mistakes in the future.
First, pBA002a_rev. It appears like mixed DNA? But only up to something like 294 nt, then it looks OK. But when I BLASTed that sequence, it's E.coli chromosomal DNA. So I suppose my plasmid was contaminated by E. coli chromosomal DNA and it happened to contain sequence similar enough to my primer? But why would the mixture stop after 290 nt?
Second are gAtNSH1_S_7_1 and gAtNSH1_T_6_1. These are from cloning the same gene, but different primers (with/without STOP codon) and independent colonies. But if you check around position 782/783, they look very similar. Before that position, the intensity is around 1500, while very shortly after that position, the intensity is only around 500. Moreover, this specific position - the first G in TGGT - is actually T to G mutation. But it is already in the plasmid, so I wouldn't expect a mutation there. Plus it's the same in two independent clones. Any idea, what could've happened here?
Thank you.
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Reverse sequence. If this is sequence from PCR amplification then I would expect that there is a small unwanted amplimer of about 290 bases so you are sequencing 2 sequences for 290 bases and this means that the sequencing mix is getting used up so that the signal strength at 600 plus bases becomes weak due to lack of reagent. Using more abi mix should give a longer sequence and stronger but a more stringent pcr annealing temperature may be needed to eliminate the unwanted amplimer.
Forward sequences. Excellent sequencing and most people would be happy to get 780 bases of good sequence. Since the intensity drops but the quality of the sequence does not degrade I think that you are diluting your sequencing mix to less than the 8ul recommended by ABI ( as every sensible researcher does) but it may just be that you are running out of reagent after such a long extension and I expect that if you are using 1ul of sequencing mix per reaction then using 2ul would give you a stronger signal for longer
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I have introduced 1 base mutation which cause 1 amino acid changed by PCR then transformed this plasmid to DH5-alpha. At this step, I checked desire transformants byspread on selective media (LB+Amp) and picked up some colony for DNA sequencing. After I got the right point mutation plasmid, I transformed it to yeast using auxotrophic marker (SD without Ura). I've got lots of yeast transformants, this come to my question, how can i make sure that my desire plasmid is transformed to the yeast cell. Another question is about the molecular knowledges that my yeast transformant contains its original gene and the mutate gene from plasmid, how can i determine that this mutate gene is affected to cell metalism since there is another original gene in the cell.
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Hi,
I will recommend you use a yeast mutant for the gene you mutated. That way, you can easily select your transformants and can determine their cellular consequences of your mutation.
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I would like to know how to get all possible DNA sequences that encode every 20 amino acid (aa means 60nt) frame in protein X and all possible aa substitutions at each residue of this protein.
I want every 20 amino acids in protein X to be linked with 10nt-barcode (huge library, 20 power20) and then test the expression of this protein using high-throughput sequencing. I would like first to get a few sequences of this system. If this works, I will order libraries that combine barcodes-10nt with protein X-60nt and then test the whole library expression using deep sequencing.
The sequence I want looks like this before cloning it to the vector:
NNNNN-[Barcode]--XXX--protein X-60nt(20 aa)]-NNNNN
XXX are 3 restriction endonucleases.
Please help me out with any available tools or websites to do this.
Thank you
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If that's your project, to make billions of plasmid vectors to expression 20 amino acid combinations & screen them for an unstated "something", my advice is to drop this project and run.
Doing a 20 amino-acid "sliding window" approach to chop up a protein is NOT the same as taking a given 20 amino acid sequence and swapping out all of the residues. You can't be serious about trying both together.
Either there is something that you are not sharing because you are worried about leaking confidential information or you are fundamentally not understanding the project or this project is a huge waste of time and resources.
If you are working on the COVID19 spike protein, there are LOTS of labs studying it's ability to bind human cell receptors (that's my guess based on your vague description).
Talk with your advisor. You say this project is funded. There is no way it's a "let's just make billions of plasmids & see if they do something" proposal.
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With Thermo-Fisher no longer providing support in the form of service/maintenance contracts for their range of ABI 3130/3130XL DNA Sequencers, does anyone here know of another company based in Europe who can provide such services for these machines? I will also consider companies providing ad-hoc repair services.
Thank you for your time,
Dennis Ensing
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that's the problem with old but efficient machines...
you can ask at your Perkin Elmer representative, maybe he'll give you a solution. it must exists.
all the best
fred
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Hello Dear, Scholars and scientists, Does PCR identify the bacteria at species level using primers without using DNA sequencing machine?
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You should be able to do this with a multiplex PCR where you use primers that are specific to a conserved site in a particular lineage but which has sequence differences between lineages, resulting in bands of different sizes. Another alternative would be RFLP analysis following PCR which can also be done using just a gel, assuming the amplicon has restriction sites in the gene region you amplify which varies between lineages. Another approach would be a genotyping qPCR where you have lineage specific probes - but those can be tricky to design and comparatively more expensive. Is there a specific type of bacteria you are working with?
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I am looking forward to explore / validate the role of a drug in breast cancer treatment particularly in the absence of mismatch repair proteins via in-silico analysis. Picking random DNA could not serve the purpose. Is there anyone who can suggest about the selection of DNA sequence / structure... the DNA needed shouldn't be more then 12 to 14 nucleotides and should be specific to that particular protein.
I am trying to dock the drug with DNA (with or without MMR proteins).
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if you are looking for nucleotide sequence... plz go to protein database .. if you want normal sequence ... if you want mutated then OMIM or other ... and wanna compare , i think BLAST is the best tool for comparison
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Hello! I am trying to do a new report on happiness and how it is embedded on our DNA. I was wondering if anyone knew the DNA sequence for happiness? I am trying to compare how happiness looks like in a person that is not diagnosed for depression and how it looks in a person that does have depression.
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The following RG link is also very useful:
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I am looking for DNA binding sites for transcrptional regulators in bacterial promoters.
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Thanks Luisa. I do not know if those are the kind of tools I am looking for, but it is quite interesting! I am going to check if they include any functionality I could use.
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I am in the process of sequencing an antibody using DNA sequencing in addition to de novo protein sequencing using mass spectrometry. When comparing the results I seem to have differences between the two results. Is this common, and are there any explanations for this result?
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How did you translate? Is size of amino acid sequence different? What is your model? Did you use the correct translation code?
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I know that several genes come one after the other under a single promoter in an operon, but what is exactly between those genes? Does the start codon of the second ORF come right after the ORF of the first gene? If there is a specific example with the dna sequence, that would be great.
Also, does an operon always require an operator?
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ORFs of eukaryotes are typically different from those of prokaryotes. In eukaryotes, mostly, genes do not come as long ORFs; they are "mixed up" with introns. Thus, providing substantially large spaces for non-coding genes (which has been demonstrated to be as high as 62% in the human genome). In bacteria, there is relatively very low non-coding DNA in the genome (approx 11%) which therefore gives in a continuous ORFs. What may be located between ORF's of two different genes, or the intergenic regions may comprise of the list succinctly put by Dr Frank Burns. It is also however, important to remember that some overlaps do occur that complicates the assessment in a single shot.
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Hi,
I am interesting what is in bioinformatic most requested from companies and academia to find easiest a job? DNA sequence analysis or? Can someone suggest that if you know how to work with something would be in moest companies requested and have good chance to get a job? and is in industry and academia same situation, or is acadamia are different things needed?
What you suggest?
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After DNA sequencing, I have to perform proofreading and quality trimming of forward and reverse DNA sequences that will be assembled to come up with the full length of the amplified DNA fragment to ensure only high-quality data is used for downstream analyses such as BLAST analysis and phylogenetic analysis.
So my question is how to assemble high quality reads of the forward and reverse DNA sequences using codon code aligner or any other software?
Thankyou
Regards
Zulaykha Khurshid
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If not, what adjustments need to be made? Thanks.
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Thanks for your reply, Tom. We want to do both single-cell DNA and RNA sequencing using the same batch of cells (dividing them into two groups, one group for DNA and the other for RNA). We've already had a plate-seq protocol for single RNA-seq. It would make our process easier if we can adapt our single RNA-seq protocol for DNA sequencing.
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I have several PCR products about 400-500bp length. How should I fragment these PCR products to get 250bp DNA? I haven't any restrictases and have only Covaris sonicator, does it fit well for the fragmentation of so short DNA sequences? I utilized it before to fragment genomic DNA but I feel some doubts about its capability to fragment so short PCR products.
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The main reason being that the quality of the base (confidence with which a photo or chemical signal can be interpreted into a nucleotide identity) decreases with length and after a point it becomes hard to identify the actual base or nucleotide call Denis Lagutkin
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I have a problem and I don't know how to answer this question. I did RLFP PCR in patient X for the presence of a specific pathogenic mutation. I got no mutation. Since the patient looked sick, DNA sequencing was done. In sequencing, the mutation came out. I would like to add that the patient was repeated with both the first and the second method. At the same time, controls were made in which the mutation tested had emerged in RLFP PCR. This was the first time I had encountered such a situation. How to explain it. I would like to add that it is a heterozygous mutation.
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If the amplification primers for the sequencing PCR and the RFLP PCR are two different pairs, and the patient is heterozygous by sequencing, then the issue may be a sequence difference that affects the binding of the primers for the RFLP PCR on the gene copy with the mutation. If this were the case, then your RFLP PCR would "work", but the only product would be from the normal gene copy. Does your sequencing cover the region where the RFLP PCR primers are located? Is this mutation one that you are following in one family or does it occur in many unrelated people?
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I am working on g-qudruplex dna sensor for that i need to know how to calculate the concentration of ct-DNA as i have to show the selectivity of different dna sequences.
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Thank you Mr Malina for your answer. But i have a question regarding what u said for the extinction coefficient of calf thymus DNA. My research lab just purchased CT-DNA from ROCKLAND supplier and another researcher who used the same supplier used a extinction coefficient of 13100M-1cm-1, is the extinction coefficient different for each supplier of CT-DNA?
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I have treated some of my plasmid samples with RNase A to get rid of their RNA contamination.
I'm going to send the samples for sequencing afterward.
Is it necessary to clean up my samples after RNase treatment?
Does RNase A make any problem in the DNA sequencing process?
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Yes, you should remove the RNase from your sample.
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Does the orientation of relatively short (eg between about 40-100bp) bait DNA sequence in a Y1H assay matter that much? Or is it very much sequence-transcription factor dependent?
The transcription factor I am using is fused to the GAL4 AD at the c-terminus, so I am wondering if the orientation is important, then the GAL4 may be pointed away from the reporter gene start site, thus reducing expression.
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The length of 40-100 is good and using to oriented it in c terminal is right and that will help you in you assay.
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The motivation behind this question is to understand how researchers discover new retrotransposons in humans?
Thanks in advance for the help.
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Searching the sequenced genome is the best start before moving into wet lab work. Different software/approach are scattered around in literature.
Here is a good start:
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Hello dear researchers
Would you please introduce a Widely used and suitable database for protein, carbohydrate, RNA, and DNA sequences?
The purpose of my model is to predict protein with peptides, DNA, RNA, and carbohydrates
Thank you
I am waiting for your guidance
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Also check EMBL
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We plan to carry out a RNAseq project with BGI but before we proceed I wanted to know if anyone has used this service and if there were any issues one had with it.
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Thank you for your responses :)
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With advancing science , more and more higher throughput technologies are available. Any one lab cannot claim to be expert in all technologies. To remain updated and learn better techniques, a student has to attend workshops and hands on training in labs proficient in such technologies. Array CGH, next generation sequencing, proteomics and metabolomics are now coming up. The brighter and sincere students can opt for these advanced technologies for their PhD work. Sometimes, some private laboratories make research work done in more easier way. However, for the benefit of the student, and also for his/her future career; the person should learn few of the best technologies available in the country or around the world. Any suggestions from your experiences?
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luis alberto mendez-rosado Well said. Older techniques were cost-effective in absence of sophisticated technologies. A problem with newer technologies with sophisticated and expensive platforms is that once the machine is out of order, work stops. The lab may not have a backup in-case of expensive equipments. Also older technologies involved more of technical expertise which improves further with experience as the students have hands on training on it and use it on regular basis.
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I have been working on recombinant p53R248Q and p53WT. It is necessary to show that the proteins are functional. For the wild type, I can show that the protein specifically binds to cognate DNA sequence. For p53R248 this doesn't hold true because the mutation abrogates DNA binding activity. So how do I show that p53R248 protein that we expressed and purified is what it is from a functional point of view. Is showing loss of function compared to the wild type protein enough?
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You can do an EMSA (Electrophoretic mobility shift assay). It is a common technique used to validate protein-nuclei acid interactions. You can amplify the cognate sequence and assay the binding efficacy of p53WT vs p53R248Q.
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is there, by any means, we have the full complete 100 million nucleotides of the DNA sequence of humans?
I want to know where can i find the DNA library which have our DNA and other DNA sequences
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The NCBI website has many useful applications,programs and databases and useful information about how to use all of the applications
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I have a list of almost 3000 DNA sequences where each sequence is separated by '>'. Using BioPython I can convert each DNA sequence to its corresponding mRNA transcript by giving input manually. But as I have a large number of sequences to work on, it is a very lengthy procedure. Is there any option to automate the input procedure so that It will automatically take input DNA sequences from the file itself?
Thank you in advance.
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just modify your Biopython script. Use a for loop :)
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The literature says it is around 2 for DNA sequencing data. Does value below 2 mean problem in sequencing?
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The ratio of transitions to transversions is often far greater than 2 for closely related organisms. For more distantly related organisms it is usually less than 1. So you really need to consider both the total pairwise distances being compared and the pairwise transiton:transversion ratio observed. The DAMBE phylogenetic package makes nice plots like the two I am attaching here.
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For identification of Bacteria, 16S DNA sequence is important. I would like to know whether partial sequence would suffice for it or complete sequence provide better clarity. in addition to that some time partial sequence matches with the some part of large sequence of 16S DNA of bacteria in that scenario how can we determine that the whether bacteria is novel or not
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16S rRNA gene sequence length of 1200 bp or above can be used for the identification of bacteria.
If the sequence similarity is below 97% then it can be considered as novel but as said 16S rRNA gene sequence alone can't determine the novelty.
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I want to go more on my analysis on DNA sequence, I want to cover all properties that nucleotides may have, so what about charge property, could it be available by default on bases, or DNA sequence needs to pass through current to get charged and by which rules the charge is distributed among nucleotides, and whether the charge is per base or for all DNA sequence?
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I think an important issue here with respect to charge is that isn't just about the DNA. DNA is important because it is an electrical conductor, something you don't see in sequencing models. However, Not all causality lies at a genetic level. Perhaps even more important is what happens at a molecular level. Cell membranes can be polarized or depolarized. The importance of membrane charge is its effect on function. Embryonic and cancer cells (i.e., motile cells) tend to be depolarized whereas differentiated cells are hyperpolarized. Michael Levin's group at Tufts has also shown that this can happen posttranslationally so that sequencing will not identify it, despite the fact that it can have a major effect on function.
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I have found several eukaryotic promoters/genes. However, if I wan to express a sgRNA in E. coli, it should be possible. Am I wrong?
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Thank you Niklas. I am sure I have to dig deeper.
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I want to encode Network packets (Commands & Attributes) using DNA sequence, but I want to map those network commands into a meaningful featured DNA sequence by the meaning of finally when you see the encoded network command, each codon has logical relationship with successive one, beside each codon needs to refer to existing Amino Acid.
Can we build DNA sequence that contains features rather than just letters (A,G,C,T) sequenced randomly beside each other? Does this have reference in Biology, what is your thought in this?
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George Church recorded an entire book, including illustrations, as a DNA sequence. Perhaps you can use his approach.
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After the SDM (Site-Directed Mutagenesis ) on a sequence of the protein from Myxococcus xanthus, then cloned in E. coli DH5 alpha, the sequencing results showed a long region disappeared (1807bp). However, comparing with the WT plasmid on the electrophoresis gel, the length of DNA looked similar (a little bit higher than the WT).
I am considering the repair mechanism of E. coli. Did you guys meet the same problem on your bench? Could you give some paper that relates to that kind of problem? Or how to check it on the bench?
Some information and clues:
The GC contents of this sequence are high because of M. xanthus.
PCR thermocycling condition (annealing at 60℃), and using Hot Start polymerase to lead a misbinding.
Contamination will lead to that happen or not?
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Thank you, Adron.
I've sequenced again and just got the sequencing result. It showed around 1800bp missing again. I forgot to mention that I didn't get the results by using forward primer both times.
So, I think you are right. Probably because of the mess of mutagenesis.
BTW, I've used the nanodrop to check the plasmid quality, showing all right (concentration and A260/A280).
I appreciate your help!
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What is the cheapest 'Next gen DNA sequencer' in the market?
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yes, as Katie A Burnette said, since you're new in this area which needs high technicality materials and skills, maybe you should send the samples to be sequenced and analyzed to a private company such as genwiz, qiagen, illumina...
stay safe
fred
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Hi, Can anyone suggest me where to find the genomic DNA sequence for K562 leukemia cell line.
I am interested in finding out the DNA sequence at the BCR-ABL junction.
Thanks!
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Hi Nidhi
looking in GEO you'll find enough data on K562 cells (https://www.ncbi.nlm.nih.gov/search/all/?term=K562) and particularly the sequences of interest (in nucleotide option).
all the best
fred
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Provided that we have a double helical DNA sequence (5-100 no) with internal Fluorophore and quencher tag in the sequence, is it possible to successfully clone the sequence in a vector?
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Could you be more clear about 5-100 no? DNA sequence upto 2kpb can be easily cloned to any vector. Just amplify the whole sequence with Digestion sites (specific to MCS) in the fwd and rev primes.
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I want to collect and preserve insects during a 2 weeks field trip, for later extractions of high molecular weight DNA for long-reads DNA sequencing (PacBio HiFi & ONT). What would be the best way to preserve the samples? Thanks a lot. Charles
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Dear Charles, many thanks for asking this very interesting technical question. As an inorganic chemist I'm certainly not an expert in this field. However, I just came across the following potentially useful article:
Utilizing field collected insects for next generation sequencing: Effects of sampling, storage, and DNA extraction methods
The paper is Open Access and it has been posted as public full text on RG. Thus it can be freely downloaded as pdf file. The authors suggest netting of the insects directly into 100% ethanol.
Good luck with your work and have fun on your field trip! 😎
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I am working on a project to classify DNA sequences using Deep Neural Networks (DNN). However, the DNA sequences are of unequal length, and I want the input sequences to be of the same length before converting them into numerical form to prevent an imbalance of training. What is the best way to convert input DNA sequences of variable lengths to equal lengths to increase the accuracy of prediction?
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You could add a state register. A gliding window goes over the DNA sequence, and at each step, the register takes the output vector of your primary classifier as input, along with its own output, maybe some additional set values, and updates its state (= its output).
Regards,
Joachim
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Hi,
I am planning to finish my PhD at the end of this year and need to express proteins from a DNA sequence. 3 out of 4 DNA sequences could be expressed without problems, but not the fourth. The insert is about 1,500 bp size and is in pet26b(+) vector. In the meantime, I have changed the peptide sequence (or the DNA sequence) three times and have also tried to carry out the expression at 30°C instead of 37°C. I use Bl21 bacteria for expression. Can anyone tell me another bacterial strain and / or expression vector that I could try out? I would be very grateful for further suggestions for improvement and possible solutions.
Thank you very much and best regards,
Christian
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In addition to the above suggestions, please consider whether the target protein needs to be co-expressed with chaperones or other proteins that form a complex?
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I have the sequences of mtCOI region and I want to convert it into a colour coded barcode for better presentation. Does anyhave have the idea on how to achieve this?
Your help is appreciated
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Thank you Dr.Hossam
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What would be the cause of a smeared bacterial gDNA band? Could not changing tips between loading a negative control after Loading DNA sample lead to a faint band of same size ( just trying to find the cause of the faint band since no reagent was contaminated)? what can I do to improve my gel.
1 % gel 95 V for 60 minutes 1kb ladder
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It might be due to migration differences of high MW gDNA.
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I have Brassica napus transgenic plant sequences and wild type sequences. I want to see whether they are homozygous or not by looking at chromotograms.
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I might be too vague, but with a chromatogram look at the differences. In any case cross them with both recesive aa.
RILS and ILS (interbred lines especially)
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Hi everyone,
I am trying to sequence a plasmid construction using the primers we used to obtain the insert by PCR. Primer has a Tm of 58. After trying with an annealing Temperature of 50 I obtained the image attached. It seems to work correctly but suddenly the detector saturates for the 4 dyes. It seems there are good peaks just before and after this, but the reaction stops too soon. Any idea of what is happening?
I have reinfected the reactions but I obtain exactly the same
Thanks everyone in advance
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Someone else may be able to diagnose it better than I can, but to my eyes this is a failed reaction pretty much the entire length. So its not like it started well and then failed.
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I'm using a multistep approach to insert multiple genes in sequence into the pHIV lentiviral vector. I am PCR amplifying the genes from either gblocks from IDT or other plasmids. I use PCR to amplify the genes of interest, confirm their size by agarose gel, and then attempt to assemble them using the NEB HiFi kit (specifically the 2x NEB HiFi master mix). When I submit the assembled plasmid for sequencing I am able to confirm insertion of the gene of interest for that round of cloning. However, the problem I am experiencing is that after several rounds of inserting each gene, I have gone back and attempted to sequence the entire assembled cassette and am noticing there are random sections of gene sequences missing.
Has anyone else seen this? Does anyone have any suggestions for working around it? Is there a better kit available?
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Hi Nicholas,
Never had problems with NEB HiFi. The source of your mutations is most likely PCRs, oligos or gblocks. Using Sanger sequencing results, determine where mutations tend to occur.
If it's in the overlapping regions of your assembly, this is most likely due to poor oligo quality. If it's within the fragments it can be due to poor PCR fidelity, use a high-fidelity polymerase (I'm using Phusion or Herculase II).
If you are already using a high fidelity polymerase and still observe a high rate of unexpected deletions/mutations outside of the oligo regions, these may already be present in your template gblock DNA.
Check what is the expected error rate for IDT gblocks, when resorting to synthetic fragments I'm using Twist gene fragments which have a error rate of 1:4000 bases. Last time I checked this was the lowest error rate for synthetic gene fragments on the market, and they were also much cheaper (0.07 $c per base).
Hope it helps
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Hi Everyone.
My query is that what are the possible ways to convert an AA sequence/nucleotide sequence into a 2D matrix so that it could be considered as an image and could be given as input to a CNN or any other image-based DNN? I have two possible solutions in mind but there are some limitations:
1. One can do it by yielding a matrix from a sequence directly by splitting it into equal rows and columns. But if sequences are of different length, the image dimensions will also vary for all sequences. Is it a good idea here to trim images to mean size or pad them with zero to make all images of the same dimensions?
2. DPC can be computed for sequences that yield a 20x20 matrix but the size is quite small. Also, it comprises information related to frequency, but the position-specific information of AA is lost.
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Hi everyone?
I'm doing my thesis about Promoter regions in some jasmonate genes on cassava (M. esculenta). I have any questions about the DNA cloninig...
Is possible lose some pair bases (bp) of the promoter's sequence?. I mean, If I have my amplified product (promoter) of 1000 pb by PCR (normal), will I get the same sequence of 1000 pb (promoter) on the DNA cloning?
Under what circumstances get lose pair bases of a sequence?
Does get lose some DNA pb being inserted on a plasmid?
On the other hand, occurs the same in the DNA sequencing?
Excuse my english (is my first post) :)
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I agree with Sylvester Ochwo.
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Hi all,
I was looking at differences between two nucleotide sequences by comparing the substitution pattern at each site. p-values for each comparison has been obtained that suggest whether or not two sequences were significantly differ from each other. There are more than a comparison for each species and I am more interested to look at the differences at species level.
As far as I concerned, p-value is more on the significant of a probability, where it tells us that whether or not the comparison was significantly differ from the null hypothesis.
The question is that is it advisable to look at the average p-value from all sequence pair comparison for each species? Will the average p-value depict the heterogeneity of each species?
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I do not think such a comparison would be very informative. If you compare the different orthologous genes in two species, you will find that some genes are much more highly conserved than others. Constraints on preserving the function of the proteins encoded by those genes limit the fraction of mutations that are tolerated by these genes. Therefore the sequence difference between two genes is not just a function of the evolutionary distance, but also depends on how essential these sequences are. To determine the relationship between remote relatives, you look at differences in highly conserved genes, e.g. histones, while for very close relatives, you look for differences in noncoding sequences, e.g. short tandem repeats in paternity testing.
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In my project, we are trying to mutate algae with artificially zeaxanthin accumulated.
I want more meaningful project, so I hope to apply bioinformatics method.
The abstract I think is if I get mutant algae with zeaxanthin accumulation, I will compare some other organism naturally posessing many zeaxanthin with that sequence.
Eventually, I can get which sequence will determine the zeaxanthin accumulation.
But it is my first time to sequence 'DNA', I am not sure how to start it.
Could you recommend me some idea or method about it??
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Hello, Lee Ha Jung.
You can use the EBI server to run some of the well-known tools to perform multiple sequence alignment.
Here it is:
Regards.
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Suggest me a low-cost DNA sequencing machine.
Please also mention the price if possible.
Thanks!
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Getting started with Oxford Nanopore DNA sequencing is easy. All products are available as Starter Packs, which include everything you need to cost-effectively perform your initial Nanopore DNA sequencing experiments. Devices start at just $1,000 with no CapEx required. But be careful with cheap products. They could make you penny wise pound foolish.
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When i submit dna sequences to NCBI, they ask for clarification saying "Some or all of the protein coding sequences contain internal stop codons, reading frame shifts (insertions/deletions based on BLAST similarity search results and/or an alignment), and/or have translations that show little or no similarity to other proteins in the database",
how to solve this problem
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if the sequence submitted has internal stop codons, then it means it's a pseudogene. There might be homologous sequences without stop codons that way it can tell which family of genes it belongs to.
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Forgive my ignorance...
I have the DNA sequence for the LCVR and HCVR of Adalimumab.
I need a resource to find the DNA sequence LC and HC Constant regions specifically Ig G1(HC) and Kappa (LC).
I tried NIH Nucleotide search but no luck.
If someone could point me in the right direction that would be much appreciated.
Thank you for your time.
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Hi My Dear,
The drug is expressed from a vector containing sequences for the variable regions obtained via phage display library methodology and sequences containing the human IgG constant domains.
You can access the HC constant region here: https://www.ncbi.nlm.nih.gov/nuccore/AJ294730.1
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I am on the lookout for the Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence. Does anyone know where I can find it?
Thank you in advance
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There are databases on GFP. Maybe you canción search in them.
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We use an AB 3130xl Genetic Analyzer for Sanger Sequencing, and on occasion, we see our plasmid samples struggle with wavy, loss of resolution (as seen in the attached image). Our general assumption is that a contaminate is co-migrating during electrophoresis, but I'd like to know if anyone has narrowed it down to more specifics of what those contaminates might be?
I'd like to provide better troubleshooting suggestions to people on what is causing this issue. Any advice/suggestions could be greatly appreciated! Thank you.
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Thank you for the update Lisa McDougall . That is not something that I would have expected or would have thought of. I am glad that you solved the problem.
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They are mentioned in a lot of papers from the 70s to these days in many introductions (not the sequences but the viruses). However I am unable to find any sequences in NCBI.
I suppose they do not exist but I ask just in case they are in another DB or under a different name and I missed them.
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Hi Sergio
take a look at the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/?term=Avian%20Leukosis%20Viruses) where you'll find lot of stuffs on your request.
stay safe
fred
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Trout and other salmonid taxonomies are still in a chaotic state and in many respects have advanced little since the 19th Century. Salmonids are renowned for their phenotypic plasticity expressed under different environmental conditions. This high plasticity in many morphological characters and life histories is such that almost any population will be found to differ from other populations especially if only a few populations are compared. Yet such characters are the basis of many species descriptions. Some claim to be following the Evolutionary Species Concept (ESC) of Simpson (1951), where “An evolutionary species is a lineage evolving separately from others and with its own unitary role and tendencies”. Evidence for the ESC is provided by morphological differences that are adaptive in nature (my emphasis), i.e., by definition have a genetic basis (Simpson, 1961). Yet many simply assume that the morphological differences that they use have a genetic and adaptive basis without further investigation even though heritability may be extremely low or absent. In that respect their approach is purely phenetic.
Since most conservation legislation is species-based accurate taxonomy is key to conservation of salmonid biodiversity. Bad taxonomy can kill by failing to recognise a population as a distinct taxon and thus it does not receive the conservation attention it requires. On the other, it can result in wasted conservation resources if the taxon is based on purely environmentally-induced differences and is simply part of a more widespread species of lesser concern. Some 51 species of Salmo trouts are currently recognised in FishBase and recent publications, including several in recent years. Most trout species have been classified on colouration, spotting pattern, occurrence of parr marks in adults, dentition, scale counts, and body measurements. In many, but not all situations, these characters are subject to environmental modulation with the effects of phenotypic plasticity and adaptation being difficult to disentangle. Body measurements are, in some cases, converted to ratios of standard length, but this approach has long been regarded as inappropriate due to allometric growth. Often insufficient specimens and populations are examined to give a true picture of intra- and inter-population variability.
An important criterion in taxonomy is that the characters used to define a species can be used to identify individuals to that species with ≥ 99% of individuals being correctly assigned (Mayr, 1963), either using molecular approaches or genetically based life history and morphological differences. Etheridge et al (2012) found that the power of supposedly diagnostic morphological characters to identify individuals of three putative Coregonus species was low (27%) due to the species descriptions being based on a few specimens, and as a result of phenotypic plasticity.
Given that a reference sequence is available for brown trout and that the determination of full genomic sequences is now relatively straightforward, is there any reason why a DNA sequence in an appropriate depository cannot be the name-bearing type sequence for a species? Linked to the type nuclear sequence should be DNA specimens, which can be used for further study. Once isolated, it can be stored nearly indefinitely. DNA can be easily shared for secure, multi-site curation. Since it takes up little space and can be stored at room temp there is no reason why all national museums should not be involved in such curation. Mitochondrial DNA sequences, while much easier to obtain, are problematic due to the potential for horizontal transfer and the linkage of genes. There are several examples of incongruence between nuclear and mtDNA. Use of only part of the nuclear genome could also be potentially problematic due to differentiation between some closely related trout being present in localised genomic ‘islands’. Sufficient DNA sequences to represent intra-specific variability would be required. Clearly international collaboration would be required to cover the entire Salmo trout range and a meaningful number of specimens. Do others consider this a potential way forward and what are the possible difficulties involved? Or is the real question whether conservation legislation should be species-based in the first place but instead be focused on populations, or groups of populations, as in North America using Evolutionarily Significant Units or Designatable Units?
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I don't think any revision of the ICZN is needed.
Regarding the morpological vs. molecular conundrum, the value of a deposited specimen mostly resides in the wealth of undescribed information that its biological structure potentially contains. We simply don't know what further information can be gleaned from a preserved specimen.
Therefore, no improvement could come from replacing a part of this structure with another. Tissue samples for molecular analyses and genomic sequences can be simply added to the lot including the morphological specimens (that could also be preserved with different methods; e.g., both formalin- and ethanol-fixed), as well as any other documentation (e.g., photos of freshly dead or anaesthetised specimens, morphometric measurements previous to fixation, X-rays, ecological notes, environmental data on the habitat). This is already common practice in several large museums, though it is obviously left to the individual researcher depositing the lot. Encouraging this practice is certainly commendable.
Therefore, I would not see any improvement in replacing morphological specimens with genetic/genomic sequences. On the other hand, I think that a considerable improvement may come from preserving and depositing morphological specimens of populations that genomic analyses have demonstrated to be reliable evolutionary units. The deposition of such material, both morphological and molecular, could certainly be a strong basis for defining a taxonomic and/or conservation unit, something the conservation biology of salmonids urgently needs.
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To perform the Luciferase assay for microRNA-gene interaction its necessary a primer (wild-type) to amplificate the specific ligation site and also a mutated primer. But if your primer has a mutated sequence, which will not recognize your DNA part, how to obtain the DNA sequence to insert in the cloning vector?
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You can use kit or other techniques.
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I have an inducible expression of the enzyme in a newly derived bacterial strain, and methylation may be affected. I want to test methylation efficiency with a bisulfite kit. The kit is the EZ DNA Methylation kit (Zymo Research, D5001).
Do I need to make a special plasmid with a certain sequence for the bisulfite test, or it works more or less equally for any DNA sequence?
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Dear Joshua Beytebiere , thank you for your kind reply! I was not precise enough in my question, and my wish to keep it short played a bad joke.
I just want to test the methylation state of the DNA in my strain roughly. My plan is:
1. to make a plasmid with a certain sequence insert
2. clone it into a plasmid,
3. transform into bacteria,
4. stimulate expression of my gene, that affects methylation
5. harvest plasmid,
6. make the test with EZ DNA Methylation kit (Zymo Research, D5001)
7. Send 20 clones for Sanger sequencing of the insert.
So I was looking for a special sequence for the insert, that would be my template to test the methylation with bi-sulfite. from your reply I guess the sequence of the "bait" doesn't matter. Thank you!
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I am writing an article about a molecular survey on viral poultry diseases. I want to compare several sequences belonging to the same virus but different in pathogenicity and at molecular level so I need to choose, align, make phylogram for viral sequences isolated by our study and ones published in Genbank. I want to know principles of aligning sequences for e.g. possibility of aligning short and long sequences? When to delete gaps in the sequences?
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Thnaks guys for answering my questions
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I have a clone of DNA sequence encoding the full spike protein (S1+S2)
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In my opinion, the longer the polypeptide, the more potential b and t cell epitooes, so I would focus on full length spike sequence.
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I am actually need to know whether there is anyway in India to file a complaint for giving fake DNA sequencing results
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In procedures for extracting DNA in the laboratory for sequence analysis and molecular studies, there may be problems with contamination in samples, methodological errors, or confusion in the identification of samples resulting in false positives or imaginary species. This is seen a lot in phylogenetic analyzes that do not make sense and are still published in prestigious magazines.
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Hello,
I have a list of Ensembl protein Ids ("ENSP...", got them from PAXdb) and I wish to find their matching dna sequences.
It seems trivial but I didn't find a way to do it...
I could find the appropriate gene Id for each protein and then get the cds nucleotide sequence but it seems inaccurate (because of alternative splicing).
Any thoughts?
Thank you!
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Use Ensembl Biomart.
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Dear researcher
I am preparing the technique for mutation detection. It has many DNA sequences, this gene has 51 exons, and we want to detect whether any mutants exist over the sequences. What is the best approach to do that, in terms of cost and time. Any suggestions will be highly appreciated.
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As it is a rare disorder you cannot be entirely sure that only one gene causes all cases of the disorder. NGS does have the advantage of finding changes in other genes but Katie A Burnette is right that the analysis is expensive and complex so you may either need a contact who has the analysis software and expertise to help with this aspect or at least a very detailed knowledge about what analysis the company doing the ngs will provide and whether it will cost extra or is built into the cost of running the sample
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Hi Everyone,
I am trying to sub-clone TCF7 gene (transcript variant 1) into pCDH-CMV-MCS-EF1 Puro vector from some other commercially available vector by InFusion cloning(recombination). I got nice appropriate size specific band during PCR amplification, the positive colonies, I send it for sequencing and when blast I could see the match in DNA sequences. Interestingly, DNA sequences match from start to around 150 bps, then around 200 bps is missing and then rest all match up to Stop Codon. Could you anyone please suggest me why DNA sequences are missing from the inbetween the gene from the clone.
Thanks!
Abhishek
Search Results
Web results
Vector Database - pCDH-CMV-MCS-EF1-Puro
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I'm seeing a lot of poly G and poly C regions in it, and I'm guessing that could be what the problem is, if the missing ~200 bp chunk is flanked by regions with a lot of C/Gs. Homologous recombination based cloning methods like InFusion and Gibson Assembly are prone to misassembling repetitive sequences.
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PCR cloning kits allow to clone a PCR product in a variety of vectors through a simple blunt-ended ligation.
However, would this still work with PCR products containing modified ends (obtained with modified oligos such as biotinylated of fluorescin-labeled primers) ? I only need the DNA sequence to be inserted into the vector, not the label.
Thanks
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Tried it and it worked perfectly fine with 2 different modifications (biotinilated oligos and fluorescin-tagged oligos) ! I have the same amount of transformants as another blunt cloning with regular (non-modified) PCR products.
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Recently I faced a problem while separating 3 species of genus Lethe butterflies (group Woodbrowns -Satyrinae) working along an elevation gradient and different forest habitats in Kedarnath Musk Deer Reserve in the Western Himalaya. Based on wing morphology I separated them into 4 species out of 5 known from the region, but examining of male genitalia revealed only 3 species while the rest were all hybrids!!! Can DNA bar coding help in identification of hybrids and their parents?
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Thank you Ajay, Ali & Md. Mizanur for your valuable comments.
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We have a PCR product that is approximatly 350 pb, and we want to see if there are more DNA sequences withn that product. We decided to use a polyacrilamide gel, but its the first time we ever do one and no one else has done it in the lab before. We don't know what will be the recommended voltage for running it and the approximate time. We did a first try and let it for 16 hrs at 50 mA, since thats what the manual said but it is made for proteins. Any suggestion will be appreciated.
I adjunt the gel image, its messy.
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I am lay in this subject. Is DNA sequencing the best way to identify the different types of soil bacteria present in soil samples? Ideally, I'd like to find a technique that allows me to identify all different types of bacteria (from those that could be identified) present in a given soil sample. Thanks.
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Soil is a rich repository of microorganisms. Basic techniques include isolation by either pourplating or spreadplating on appropriate media. Identification can be done by laboratory procedures such as gram staining, motility test, spore staining and a battery of biochemical tests. Quicker results can be obtained by 16 S r RNA sequencing technique. Comparison with NCBI gives similarity of your organism with others.
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I would like to design primers that are complimentary or begin in the UTRs (5' and the 3') of a gene? Where exactly and how do I find the coding sequence that includes the UTRS ?
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You can find the gene coordinates using NCBI then you can try to design primers on the near outside regions.