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jerryshelly1's Content

There have been 197 items by jerryshelly1 (Search limited from 16-November 18)



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#169223 Protein extraction from RNAlater

Posted by jerryshelly1 on 07 July 2014 - 09:46 AM in Protein and Proteomics

Googling brings up a plethora of detailed article and notes from the manufacturer...

 

http://www.ncbi.nlm....pubmed/12459147




#169073 SNPs analysis

Posted by jerryshelly1 on 01 July 2014 - 07:22 AM in ChIP and Next Generation Sequencing

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




#169037 orthologs of genes

Posted by jerryshelly1 on 30 June 2014 - 08:09 AM in Cell Biology

http://orthodb.org/orthodb7




#167907 Protein Glycosylation and SDS-PAGE

Posted by jerryshelly1 on 19 May 2014 - 05:21 AM in Tissue and Cell Culture

How are you extracting your protein




#167836 Conservation of SNPs contexts

Posted by jerryshelly1 on 15 May 2014 - 11:32 AM in Bioinformatics and Biostatistics

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.




#167750 How Dorothy Hodgkins google doodle is read

Posted by jerryshelly1 on 12 May 2014 - 09:38 AM in Chit Chat

I'm in America and we have the Hodgkin doodle.




#167747 How Dorothy Hodgkins google doodle is read

Posted by jerryshelly1 on 12 May 2014 - 09:07 AM in Chit Chat

very neat!




#167742 What brand of gloves do you prefer?

Posted by jerryshelly1 on 12 May 2014 - 07:44 AM in General Lab Techniques

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.




#167417 Is this primer-dimer peak in my melting curve?

Posted by jerryshelly1 on 30 April 2014 - 09:45 AM in PCR, RT-PCR and Real-Time PCR

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...




#167408 Is this primer-dimer peak in my melting curve?

Posted by jerryshelly1 on 30 April 2014 - 06:00 AM in PCR, RT-PCR and Real-Time PCR

Are the peaks at 76C and 80C from the same primer set?




#167405 Is this primer-dimer peak in my melting curve?

Posted by jerryshelly1 on 30 April 2014 - 05:12 AM in PCR, RT-PCR and Real-Time PCR

I don't think you showing the melting curve for a primer dimer. If you look at each sample individually do you see a little peak near the left of your plot?

Attached Thumbnails

  • post-6817-0-14729200-1398844686.jpg



#167404 If I am running Real-Time PCR with one gene of interest and one reference gene(G

Posted by jerryshelly1 on 30 April 2014 - 04:59 AM in PCR, RT-PCR and Real-Time PCR

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?




#167351 My beta actin bands are changing

Posted by jerryshelly1 on 28 April 2014 - 05:32 AM in SDS-PAGE and Western Blotting

just curious... What does your cell look like when you reach the drug concentration that results in a loss of your beta actin bands?




#167297 how much purify protein nedded for IP

Posted by jerryshelly1 on 25 April 2014 - 06:01 AM in Molecular Biology

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.

 

http://www.abcam.com...ource&rid=11385




#167270 how much purify protein nedded for IP

Posted by jerryshelly1 on 24 April 2014 - 09:17 AM in Molecular Biology

Consult the data sheets for your antibody and/or purified protein (assuming you purchased them). They usually indicate the amount to use for these type of assays.




#167266 My beta actin bands are changing

Posted by jerryshelly1 on 24 April 2014 - 06:41 AM in SDS-PAGE and Western Blotting

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?




#167257 My beta actin bands are changing

Posted by jerryshelly1 on 24 April 2014 - 05:30 AM in SDS-PAGE and Western Blotting

What does the drug do? Is it a cytoskeletal drug that targets structural proteins?




#167256 Detection of mutations in plasma samples using multiplex amplicon sequencing

Posted by jerryshelly1 on 24 April 2014 - 05:24 AM in ChIP and Next Generation Sequencing

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.




#167170 NGS of bi-partite viral genome

Posted by jerryshelly1 on 21 April 2014 - 06:44 AM in Microbiology

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.




#167169 ChIP Buffers

Posted by jerryshelly1 on 21 April 2014 - 06:30 AM in ChIP and Next Generation Sequencing

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.

 

http://docs.abcam.co...ide-to-ChIP.pdf

 

http://www.millipore...ech1/tp5994en00

 

http://www.promega.j...chap6.pdf?la=en




#167134 I have a careless boss

Posted by jerryshelly1 on 18 April 2014 - 06:19 AM in Venting and Counseling

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.




#167037 restriction enzymes/sites to avoid

Posted by jerryshelly1 on 14 April 2014 - 01:06 PM in Molecular Cloning

Weird overhangs such as AleI that produce weird cut sites with "any" nucleotide. I just try to avoid using these all together.

 

Ale-I-cutsite_1_v1_000007.gif

 

Edit - I guess they are not necessarily weird, I just prefer not to tinker with them. I like my common REs.




#166962 restriction enzymes/sites to avoid

Posted by jerryshelly1 on 11 April 2014 - 11:20 AM in Molecular Cloning

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)




#166953 website for primer design

Posted by jerryshelly1 on 11 April 2014 - 05:51 AM in PCR, RT-PCR and Real-Time PCR

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.

 

http://www.plosone.o...resentation=PDF

 

Good luck!




#166926 PCR not working

Posted by jerryshelly1 on 10 April 2014 - 10:18 AM in PCR, RT-PCR and Real-Time PCR

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.





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