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jerryshelly1's Content

There have been 197 items by jerryshelly1 (Search limited from 01-June 19)



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#164209 Lymphocyte Cell Line

Posted by jerryshelly1 on 16 January 2014 - 09:12 AM in Tissue and Cell Culture

Hey guys,

 

I was wondering if anyone has worked with a lymphocyte cell line called AJ38? I saw the cell line referenced in an old paper, but I cannot find any relevant information about it through Google. It appears as though everyone involved in the research has retired and contacting them seems fruitless. If anyone can provide any insight, it would be greatly appreciated.

 

Thanks.




#165306 WBC count in rabbit's synovial fluid

Posted by jerryshelly1 on 21 February 2014 - 10:25 AM in Animal and Zoology

I'm going to go through all the articles, but take a look at these highwire results.

 

http://highwire.stan...etype=1&x=0&y=0




#165295 Why when disigning primers for RTPCR, you need to chose a region where introns a

Posted by jerryshelly1 on 21 February 2014 - 09:15 AM in PCR, RT-PCR and Real-Time PCR

Consider what your RT-PCR primers are recognizing. When you make cDNA, you are generating the transcript from RNA. By having the primers extend across the intron, you are guaranteeing that you are only reading the RNA expression levels and not any DNA that made it through your purification steps. Does this make sense? 




#164510 storage of laboratory human blood samples(whole anticoagulated blood-serum-plasm

Posted by jerryshelly1 on 25 January 2014 - 10:41 AM in General Biology Discussion

Depends what type of assay you want to do with them. We store whole blood at 4C for about 3-5 days if we want to extract DNA. Any longer than that and the blood is moved to the -80C for long term storage. The storage conditions will vary depending on what you need from the blood.




#164379 conditioned media

Posted by jerryshelly1 on 21 January 2014 - 08:41 AM in Tissue and Cell Culture

If you are only concerned about some cytokine/protein that is being excreted into your media then you should just remove the liquid without disturbing they adherent cells. Depending on what you are looking for, you may want to filter your cells to remove any of the larger particles (dead cells) that could alter your overall protein concentration. From here, you could spin down your cells and isolate your protein. 

 

Can you be a little clearer in what you are looking for?




#165830 Please comment on my Cloning Plan

Posted by jerryshelly1 on 07 March 2014 - 08:54 AM in Molecular Cloning

It looks like a very well thought out plan. Just make sure you design you primers with enough overhang for proper enzyme digestion.

 

https://www.neb.com/...agments#chart-S

 

 

Everything else you listed looks correct. Good Luck!




#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!




#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




#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.




#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.




#166162 Cloning pDsRed-Monomer-N1

Posted by jerryshelly1 on 19 March 2014 - 11:15 AM in Molecular Cloning

It would be better if you could provide a detailed description of what you are doing. There are tons of cloning resources on this website that you might consider browsing through. My most common cause of ligation failure is proper digestion of my PCR fragments. You usually get good digestion with a vector due to its size and shape, but PCR fragments can be problematic depending on the size.

 

I usually like to test the efficiency of my ligation by running a simple PCR with a primers that recognize both the vector and PCR fragment. Amplification is a good indication that some ligated products are in your sample.

 

What are your JM109 cell controls like? Do you observe a good transformation efficiency?




#163071 Primers

Posted by jerryshelly1 on 03 December 2013 - 02:23 PM in PCR, RT-PCR and Real-Time PCR

1ul Primer (100uM) in 999uL water is 100nM. 

 

1uL primer (100nM) in 999uL water is 100pM.

 

Can you figure it out from here?




#160916 Calibrator sample has zero expression of target gene.

Posted by jerryshelly1 on 04 October 2013 - 06:56 AM in Molecular Biology

Are you trying to compare the fold increase of your cytokine at time point zero vs. a later time point?

 

There are a couple of different ways you can approach your quantification depending on what you are trying to show.  Does the treatment of your sample suddenly switch on your cytokine or is your cytokine usually differentially expressed? Are you trying to compare your cytokine expression to the expression of a common ubiquitous cytokine?




#161065 ABI 7300 qRT-PCR and ddCT protocol

Posted by jerryshelly1 on 09 October 2013 - 05:59 AM in PCR Reagents and Equipments

Academic institutions keeps receipts for ages.  If ABI is not willing to show you their original purchase receipt, find your institutions.  You can also usually get some leeway if you mention that you use there chemicals, plates, films for the machine and mention that many other companies make more suitable replacements. It has work for me with some things. I guess it just depends on the overall cost of the product.




#160616 Weighing harvested cells

Posted by jerryshelly1 on 24 September 2013 - 01:26 PM in Tissue and Cell Culture

Figure out your OD. You could easily calculate "mg" of cells from this number.




#160412 Differentiating between long and short isoforms of Dopamine D2 Receptor using We

Posted by jerryshelly1 on 19 September 2013 - 10:58 AM in Protein and Proteomics

You could enzymatically deglycosylate your proteins.  It wouldn't be 100%, but you should see an relative increase in your band intensities. You could convince your reviewers on the effectiveness with any RT-PCR data you have on the isoforms.

 

NEB enzyme people have used in the lab (I'm sure others exist).

https://www.neb.com/...ycosylation-mix

 

Good luck

 

Edit-You are probably already aware, but you may be able to get a free sample to convince your PI it works before purchasing.




#160199 Protease inhibitor in wash step?

Posted by jerryshelly1 on 12 September 2013 - 09:33 AM in Biochemistry

I never add the PI at that step, just because I will be denaturing it at 95c within 30mins.  I have never had any issues.

 

Adding PI to the wash buffer following bead pulldown seems kind of excessive.  I would try them side by side and determine if the results change. At least this way if anyone higher up has a problem with it, you have evidence supporting you.




#161648 question about sequential transfection several plasmids in 293T and COS7 cells

Posted by jerryshelly1 on 24 October 2013 - 12:45 PM in Tissue and Cell Culture

How are you transfecting your cells? Are you doing a transient transfection, waiting two days and then transfecting again?

 

Do you linearize your plasmids when you transfect your cell with multiple plasmids. If not, I would at least linearize the LacO plasmid. That guy is huge and the cell may have some difficulty taking it up.

 

I would start by transfecting your lowest transfection efficiency plasmid, selecting for it and then proceeding with the others. How are you selecting for your plasmids? Does each plasmid harbor a different mammalian resistance or are you doing downstream assays to check the expression of each plasmid?




#161674 SNP in the promoter region

Posted by jerryshelly1 on 25 October 2013 - 06:49 AM in Molecular Cloning

If it is a known SNP you can go to Ensembl and see what is known about it. Another good resource is dbSNP.




#162523 free online DNA & plasmid editing tool

Posted by jerryshelly1 on 16 November 2013 - 08:47 PM in Molecular Biology Products

*Moved to a more relevant subforum.




#161802 lysis buffer for generating lysate from ovarian and brain tumors?

Posted by jerryshelly1 on 28 October 2013 - 03:25 PM in SDS-PAGE and Western Blotting

There are a lot of different detergents out there. I think you should search highwire.stanford.edu with you key words, "your cell line, cell lysis buffer, homogenization" or something along those lines. Find a good publication that has beautiful westerns and mimic their methods.




#161801 Tool for mapping references to endnote style?

Posted by jerryshelly1 on 28 October 2013 - 03:21 PM in Dissertation and Paper Writing

When you go to endnote in word > search references > the external endnote program opens > do you see a drop down menu with formatting styles? I have version X5. You can also find this info in edit > output styles.

 

Good?




#161686 GADPH does not express

Posted by jerryshelly1 on 25 October 2013 - 12:44 PM in PCR, RT-PCR and Real-Time PCR

Are any other genes being amplified from your ovarian samples? It is possible that your samples have been degraded. Try your primers with freshly extracted/prepared DNA.




#160198 Protocol for qPCR using the ABI SYBRŪ Green PCR Master Mix

Posted by jerryshelly1 on 12 September 2013 - 09:26 AM in PCR, RT-PCR and Real-Time PCR

I have used SYBR Green.  I usually used a total reaction volume of 12uL. The final primer concentration would have been 2-10uM (I played around with this and the results stayed consistent - trying to determine the minimal amount of primer I could use - check SYBR green protocol). I would add 0.5uL of a 2ug cDNAtemplate. I would add 6uL of the SYBR Green master mix.

 

My thermal cycling program:

Doublets of the reaction mixture were loaded and cycled once at 50ºC (2mins), and 95ºC (10mins); 40 cycles at 95ºC (15secs), 58ºC (30secs), and 72ºC (30secs); and a final cycle at 95ºC (15secs), 60ºC (20secs), 95ºC (15secs), and 60ºC (15secs).

 

The 58C will be unique to your primer Tm's.  I would always try to design your primer to a specific Tm.  This will allow you to perform very diverse RT-PCR's simultaneously (saves plates and optical film).

 

Good Luck.





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