Googling brings up a plethora of detailed article and notes from the manufacturer...
Jump to content
There have been 197 items by jerryshelly1 (Search limited from 16-November 18)
You could either organize them into groups or just analyze them separately. Feed your results into SeattleSeq and it will return you a spreadsheet that gives the standard dbSNP information. From there, you can compare the frequencies within the population and determine which SNPs are novel
Can you restate your question? If you are looking for general SNP information, it can be found on UCSC Genome Browser. When you search for a SNP, it will usually give you the allele frequency of that SNP in humans and sometimes other organisms. If you are trying to look at the genomic signatures, then you would probably want the raw sequencing data with the electropherograms. I think it would be best if you were more clear with your question.
I use microflex when handling more infectious materials and I use distinct gloves when performing non hazardous work. Make sure you are using the gloves before the printed expiration date. If the webbing keeps ripping, consider using the next biggest glove size.
Ok... The decrease in fluorescence can probably be attributed to a lesser concentration of product. What do your Ct values say? A primer dimer melting curve would be shifted to the left. Why would a 15bp product melt at a temperature that is equal to a 100bp product? Just run it on a gel and see what size your amplicon is. If it is the predicted size...
Maybe there is allele variability for that particular amplicon region. You could run the samples on a gel and see what the size difference is. If you see two distinct bands, you probably have allelic variation, if you see a smear adjust your cycling conditions. Are these previously established qPCR primers? Taqman?
Sure, it can be done that way. I would consider doing this if co-elution of my antibodies (heavy/light chain) had a size similar to my target protein. This method has a draw back in that it will yield less protein; however, since you are using a purified protein product invitro it may not matter that much.
Edit - Found Abcam's Immunoprecipitation protocol, 4.2 method A and B.
Its a weird result for sure. Actin is extremely stable and thus makes an excellent loading control for western blot. Maybe your drug is activating something that degrades actin? What pathway is your novel molecule supposed to be involved in?
I forgot to ask you, are you seeing a signal from your target protein? or is the the blot completely blank?
What approach are you using to normalize your samples (spec, quantitative binding, etc...). You could go to UCSC and find a common SNP and compare the reported frequency to your sample. Does your raw data indicate that you have good quality reads?
I'm not familiar with mpileup, but in our suite that we use you can adjust how each base is called to increase the number of your variants - again you can use common SNP frequencies to adjust the variant calling.
I think it gets tricky when you are doing NGS on an unknown genomic sample. Is there any homology between the two circular DNA genomes where your results could be read as a population instead of independent? I have seen population contamination with eukaryotic samples (mouse, human), but I have never done any virus work. Do you know how they sequenced your DNA?
Are you just trying to confirm what organism you are dealing with?
My favorite software to use is seqman by DNASTAR. You could fragment your sequence and feed it through seqman to see what you are dealing with. If you are trying to confirm if it is a begomovirus, you shouldn't have any problem.
Sorry if this is scattered, it is early and I forgot my coffee at home.
I found three references that look like they give a decent background on ChIP. You essentially are trying to wash away any non specific binding that is occurring. This will ensure that your downstream qPCR signal is from your antibody binding to your target.
Sorry to hear that. It is extremely unfortunate that you are in this position. How can this individual maintain her funding if she is an overall terrible mentor? It seems that the administration would look poorly on her overall negligence.
Weird overhangs such as AleI that produce weird cut sites with "any" nucleotide. I just try to avoid using these all together.
Edit - I guess they are not necessarily weird, I just prefer not to tinker with them. I like my common REs.
If you are using a manufacturer provided plasmid, chances are each RE present in the multiple cloning site has been thoroughly tested and will give decent results. I tend to avoid RE sites that produce weird overhangs or require any (N) nucleotide to cut. Always avoid compatible end producing enzymes (isoschizomers)
DNASTAR has excellent genomic software and I believe the do offer free trials. A great data mining website is highwire.standford.com. I prefer to use this website instead of Google when performing a search using keywords. I found this paper via Highwire and it looks like primers for exons 1-6 are listed.
You can't run them together. I just open two windows and compare them. If you are looking to find a common Tm, try NEB's Tm calculator.
You could always consult Emsembl or Genome Browser to see relative GC content across your gene of interest.