BioForum Bioscience Discussion http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 19:27:46 -0800 15 http://www.protocol-online.org/forums/index.php?showtopic=11705 Does anybody have any experience using this dye for staining live cells for flow cytometry? If so, would you be willing to share your protocols and instrument settings? Fri, 20 Nov 2009 15:15:31 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11705 http://www.protocol-online.org/forums/index.php?showtopic=11704
Hope you don't mind my posting a plug for my software here ... hopefully some of you will find it useful. I've written a desktop java software application called MochiView that is a hybrid genome browser and motif analysis/visualization platform. The software is particularly suitable for ChIP-Chip and ChIP-Seq analysis, and has also been used with RNA-Seq and as a stand-alone motif management system.

The MochiView website (link) contains everything you need to get started, including a tutorial and a few sample videos of the software in use (link).

Here is a summary of some of MochiView's features:

[1] Highly customizable plots… display anything from genome alignments to motif matches to bar- or line-graph data on one or more tracks. Open plots are persistent across sessions and you can save your configurations for later use

[2] The plots have smooth scrolling/zooming, lots of interactive elements, and a powerful data browser feature.

[3] Very easy to set up and use. The database is built in to the software, so no fussing around with external databases is required. You can also export your current database and plot configurations to share with other MochiView users.

[4] Motif management and analysis utilities:

• MANAGEMENT: Maintain a motif database (several libraries are available on the website to get you started)
• DISCOVERY: Find new motifs with the built in motif finder (or import MEME and Bioprospector motif searches)
• COMPARISON: Check whether your newly found motifs resemble any other known motifs
• DISTRIBUTION: Search for non-random positioning of motif matches relative to other genomic features such as start codons or instances of a different motif.
• MOTIF ANALYSIS and ROC PLOT: Determine whether your ChIP regions are enriched for a motif relative to a set of control regions
• MOTIF SCORING: Numerous options for motif scoring (LOD score, p-values, w-score) and many different export formats. For example, a few clicks gives you a "gene vs motif" spreadsheet matrix of the motif score for each gene's promoter region.
• MOTIF LOGOS: Export logos of one or more of the motifs in your library
• MOTIF GO TERM ENRICHMENT: Determine whether your motif is enriched in the promoters of genes with common functionality

[5] Location and data analysis and refinement utilities:

• PEAK EXTRACTION: Not a full-fledged peak finder (i.e. no p-values), but this utility does an excellent job of smoothing your ChIP-Chip data and marking regions of a user-defined width under peaks (a useful first step to refining a motif search).
• DATA SMOOTHING: Smooth your data and save as a "Tiled Set" (similar to a UCSC browser wig track).
• DATA REFINEMENT: Numerous filters can refine your data sets (e.g. filter by value or by overlap with other sets of locations).
• LOCATION SET SUMMARY/COMPARISON: Get a detailed description of the genome-wide coverage of one or two set of locations (e.g. ChIP binding regions)
• GENE PROXIMITY ASSIGNMENT: Determine which gene(s) lie near a set of locations (e.g. ChIP binding regions). Can be used in conjunction with a GO term enrichment analysis.
• SET OPERATIONS: Take the intersection, union, or difference of two sets of locations (e.g. find the overlap of two sets of binding regions and remove the portions that overlap ORFs)

[6] Extensive sequence search options, including direct and inverse repeats

Thanks for your time!
]]> Fri, 20 Nov 2009 14:50:44 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11704 http://www.protocol-online.org/forums/index.php?showtopic=11703
I am glad to have found this site. I apologize if this topic has already been discussed else where but I searched and could not find a specific answer to my problem.

When I began my mastermix on ice, a small piece of ice accidentally fell into one of the primers. I tried to remove the ice but couldn't and decided to continue with the the PCR reaction anyway to see what the results would be. Before the ice totally melted however, I used the automatic pipette to withdraw some primer from the bottom portion of the tube. Is the primer ruined for good and how will ice effect PCR reaction?

Please help. Any input is appreciated!

Thanks.]]> Fri, 20 Nov 2009 11:50:16 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11703 http://www.protocol-online.org/forums/index.php?showtopic=11702
Can I still digest the wells I added blue juice to? Or will I need to re-PCR that DNA?

Thanks much
]]> Fri, 20 Nov 2009 11:42:02 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11702 http://www.protocol-online.org/forums/index.php?showtopic=11701
I've got a bit of a quandary and I'd like to hear your input. My
roommate bakes bread, and I do cell culture at the lab. I know that
some labs have experienced yeast contamination of their cell lines,
attributed to baking or brewing at home.

Do you have any particular tips, other than stringent good lab
practice, that can really help prevent any contamination? I can't
prohibit her from baking (nor do I want to), but are there any special
precautions that she/we can take at home?

And from your experience: are there any of you who do bake bread at
home, without problems at work? (am I worrying overly much?)

Thanks in advance,
Megan]]> Fri, 20 Nov 2009 10:26:26 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11701 http://www.protocol-online.org/forums/index.php?showtopic=11699
I am using mitomycin c to make my MEF feeder layers for iPS cells. I ordered a 2mg vial from Sigma and resuspended in 2ml of water. I vortexed the vial to make sure the powder went into suspension completely. I aliquoted them out at 100ul each. However, after freezing I noticed that some of the vials contained blue-gray suspensions while others looked like the mitomycin had fallen out of suspension (there were blue flecks in clear solution).

Has anyone else experienced this? I have read that suspending in PBS is also done. Can I still use the aliquots with the blue flecks and hope they dissolve in the culture media? I'm trying to avoid irradiating the cells so any advice for preparing the mitomycin c would be greatly appreciated. Thanks!!]]> Fri, 20 Nov 2009 09:45:40 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11699 http://www.protocol-online.org/forums/index.php?showtopic=11698
Does anyone know the technique for hemocyte/hemolymph isolation from mosquito larvae, pupae and adults?
Any help would be really appreciated.

Thanks,
-Uma]]> Fri, 20 Nov 2009 08:09:23 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11698 http://www.protocol-online.org/forums/index.php?showtopic=11697
I want to construct a library of mutants consisting of all 20 amino acids at a specific site within a protein by PCR. I am looking for a good strategy for primer design. Can you please help me if anyone had prior experience with this?


Cheers
bio]]> Fri, 20 Nov 2009 07:39:39 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11697 http://www.protocol-online.org/forums/index.php?showtopic=11695
Hello!

I was wondering how to get labelled nucleotides, if you can buy them or make them urself (if so where can I find protocols). I know you can radioactively label them, theres also biotin....but ive been looking for a protocol or something and I can only find papers where people use them. I also looked for radioactivaly labeled SAM and was unsuccesfull.


Would neone know of a protocol or company and maybe pros and cons of using labeling?


Wish you all a great weekend!]]> Fri, 20 Nov 2009 06:27:45 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11695 http://www.protocol-online.org/forums/index.php?showtopic=11694
I am fighting with an Gateway PCR.The latest problem is that I pipetted 20 ul reaction last night for a Gradient PCR, using 96 well plates with that aluminum foil on top. Paranoid as I am I doublechecked that its really closed, but this morning I found less than 5 ul in each well, and of course there are no bands besides the template.

My question:
Do you make PCR reactions in those (I think Polypropylene)96 well plates and did you observe problems with that? what is different about the PCR tubes?

thanks all!]]> Fri, 20 Nov 2009 04:50:11 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11694 http://www.protocol-online.org/forums/index.php?showtopic=11693 Hi.

When I treat cells with a compound I get an huge G2/M arrest, as assessed by flow cytometry. I want to check if the cells are arrested either in G2 or in M. How can I do this?

Is there any marker I can use to check this by immunofluorescence / flow cytometry?


Thanks for any answer.]]> Fri, 20 Nov 2009 04:29:36 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11693 http://www.protocol-online.org/forums/index.php?showtopic=11692 Hi.

I want to concentrate my cell lysates for western blot by precipitation of the proteins with trichloroacetic acid + acetone. I have some questions.

1. This treatment will not modify my proteins for the posterior immunoblot detection?

2. After the precipitation, what is the best method to store the lysates? Can I freeze the pellet? Or should I ressuspend it in some buffer or H2O and store it like that?

3. Can I quantify the protein concentration by the Bradford assay (as I usually do)?


Thanks in advance.
]]> Fri, 20 Nov 2009 04:24:06 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11692 http://www.protocol-online.org/forums/index.php?showtopic=11691
I'm really unhappy with my DNA isolation at this moment since I cannot get ride of RNA from the sample, which I've heard will interfere with DNA during PCR. For removal of RNA I did RNAse treatment and then phenol extraction and CIA (chloroform+isoamyl alcohol) extraction. The DNA was precipitated using NaOAC and absolute ethanol, pelleted, washed with 70% ice-cold ethanol, dissolved in TE solution and the concentration was measured using Nanodrop. The ration 260:280 is all the time around 2. I repeated RNase treatment in same sample but no improve.
To look for the presence of other stuffs along with DNA, I also did ran agarose gel with the sample. Here is also the photo of the gel (sample and ladder). It seems from gel my DNA is not pure. I require some help, please!
http://www.protocol-online.org/forums/index.php?act=attach&type=post&id=970]]> Fri, 20 Nov 2009 03:38:37 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11691 http://www.protocol-online.org/forums/index.php?showtopic=11690 For some time now we polyadenylate in vitro transcripted and purified mRNAs with polyA polymerase from USB, but all of a sudden it stopped working.
We tried different polymerases, different templates and different ATPs. We also tried different purification methods before polyadenylation (3 Kits, LiCl and nothing at all).
Does anyone have an idea what else we could change so it works again?
Thank you soooo much.
Schnuffel]]> Fri, 20 Nov 2009 03:04:08 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11690 http://www.protocol-online.org/forums/index.php?showtopic=11689 Thu, 19 Nov 2009 23:13:34 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11689 http://www.protocol-online.org/forums/index.php?showtopic=11688 Hi

I would like to know if “non reducing SDS protein ladder” is available???

]]> Thu, 19 Nov 2009 22:48:56 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11688 http://www.protocol-online.org/forums/index.php?showtopic=11686
Anyone here has experience with hematopietic cell line expansion? I am about to buy one, but I do not know which is good..does any one has recommendation?

Past few months, i cultured cord blood hematopoietic stem cells. The problems: cells didnt expand much after 1-2 weeks upon expansion and couldnt be maintain in its undifferentiated stated. I used serum-free media and cytokine cocktails from stemcell technologies. So I was thinking, why not I try with the cell line.

Thanks in advanve for any responses..

Rgrds,
Boudou]]> Thu, 19 Nov 2009 21:23:22 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11686 http://www.protocol-online.org/forums/index.php?showtopic=11685
I am a long time fan of your forum, so I finally decided to get out of the bushes, stop lurking and actually participate in the forum discussions .

I have been reading for a long time the advantages of the fast chip protocol when compared to the regular protocols, so I finally decided to put it in practice. However I am a little confused, do I need to do the column purification (I read in the forum arquives that you use a qiagen column to purify the DNA instead of doing the phenol chlorform extraction)? And if so, which type do you use?

To clarify my doubt:

1) crosslink and harvest cells
2)lyse cells and wash pellet
3)shear chromatine and centrifuge
4)add ab
5)centrifuge to clear aggregates
6)add protein A
7)wash beads
8)add chelex and boil
9)add proteinase K and incubate
10) boil
11) centrifuge and collect supernatant
12) column purification?

Thank you so much to all of you. Your help will be greatly appreciated.
Oh, and thank you jiro_killua I learned to do the ChIP data analysis from you
]]> Thu, 19 Nov 2009 17:32:45 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11685 http://www.protocol-online.org/forums/index.php?showtopic=11684 Thu, 19 Nov 2009 16:12:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11684 http://www.protocol-online.org/forums/index.php?showtopic=11683
I am studying the function of transcription element. The experiment I would like to do is to construct a stable cell line integrated into the genomes with my interesting promoter + reporter gene and a selectable marker gene and monitor the transactivation and repression of the reporter gene mediated by the promoter upon differentiation with time. Since the promoter might be silenced upon differention, the selection marker should be under a different promoter. For method, I would prefer virus transfection, but lipofectamine transfectiion would also work. Does anybody have idea of what vector I should look for? Thank you!]]> Thu, 19 Nov 2009 16:02:46 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11683