BioForum

Using plastic for microscopy?

Wed, 11 Nov 2009 13:51:09 -0800

I am working with a cell type that differentiates very well on plastic plates that are made of polystyrene, but differentiate very poorly when grown on glass coverslips. I need to do confocal microscopy with these cells and I am not sure what to do.

I have found plastic coverslips that have the same refractive index of glass and are the same thickness, but I have heard that plastic can autofluoresce and create quite poor images. The other problem is that although these coverslips are plastic they are not polystyrene, so they still might not work for my cell type.

I have tried chamber slides (which also are a different kind of plastic) and my cells differentiate relatively well but still not great.

Another alternative is to try coating glass cover slips with something and see how that goes.

I was wondering if anyone out there has had experience with this sort of thing or can offer advice on how well plastics work for microscopy or has another suggestion.

Thanks.

BSA content in Fetal Bovine Serum?

Wed, 11 Nov 2009 13:35:32 -0800

I know serum contents vary a lot but does anybody know about an estimate of BSA content in FBS or can direct me to a source?

Suggestions for optimizing a multiplex PCR?

Wed, 11 Nov 2009 12:39:20 -0800

Hi All,

I am trying to use molecular methods to study bat diets, by amplifying insect DNA from bat feces. I am trying to set up a multiplex rxn to screen for consumption of three particular insects. I have designed species-specific primers for these three insects (a beetle, the primers produce a 495-bp product; a moth, the primers produce a 320-bp product; and a katydid, the primers produce a 250-bp product).

My primer pairs all work fine on their own (although the primers for the katydid are suboptimal and want to dimerize a bit; at this point, I haven't found a way around it while still keeping the specificity to the target). All the primers have Tm's between 55 and 60 degrees, GC content 40-60%. And, as I mentioned, each primer pair on its own works well to amplify DNA from target species and not from various other insects that I have screened.

Anyway, I would like to combine these into a multiplex rxn. I got a 100 rxn Multiplex kit from Qiagen, which uses their HotStart Taq in a master mix, and an optional mystery product called "Q solution" which does seem to reduce the production of non-specific products in my testing.

I started out by testing this by combining 50 uL from extractions of each of my three target species to make a positive control mix, and started testing out the multiplex with an equimolar mix of my 6 primers. I ran a few rxns with varying amounts of this template, at 57 C as suggested by Qiagen when one or more of the primers has a Tm of less than 60. (No gradient thermal cycler in my lab, boo hoo.) Template amount didn't make much difference, but what was weird was that I got a beetle band at 495 and a katydid band 250, but no moth band at 350.

I figured maybe I used the wrong moth extraction (i.e., accidentally grabbed an extraction from another moth I had extracted while testing specificity of my moth primer set) so I made sure I got the "right" moth, and added another 50ul of this to my master extraction positive control. I set up another couple of reactions with this template, one straight up, and one with a little extra of the moth primer (this is the one that wants to dimerize much more than the others). Spaced and ran them at 60C instead of 57C.

So now, when I ran out the product on the gel (from both reactions), the 320-bp moth product is there. The 495-bp beetle product is there. But now the 250-bp katydid product is missing... and it was there before!!!

Any suggestions on which next steps to take in optimizing this reaction? There are so many possible variables to adjust (template amount, primer concentration, annealing temp and time, yadda yadda) that I'm not sure where to begin. Or, since there are only three species I'm screening, should I just shrug my shoulders and run the reactions separately for each primer pair? Any suggestions appreciated, I'm a first-timer. I've uploaded the Qiagen multiplex handbook, which I have been following thus far.

Neurospheres

Wed, 11 Nov 2009 12:25:52 -0800

We currently work with mice brain slices to use our electrodes in order to make measurements. Though, we would like to incorporate the use of neurospheres so that we do not have to rely on mice, and instead can use our electrodes on neuron cell cultures. I only have basic knowledge about neurospheres and, as I understand, mice neurospheres are easier to do than rat neurospheres. So I wanted to just get some more information about neurospheres, various terminology associated with cultures in general (such as what are passages?), and the different methods to make these neurospheres. Also, it really does not matter to us if these neurospheres are rat or mice - we would be willing to go for the easier or the cheaper of the two.

I appreciate this. Cheers.

Software for Ligand Binding Analysis

Wed, 11 Nov 2009 11:33:29 -0800

Our lab currently uses the LIGAND program for MS/DOS to calculate Kd, Ki, Bmax, and the like. As you can imagine (since it's MS/DOS) it's very slow and difficult to enter data. Can anybody recommend something a little more user friendly?

Antibody staining

Wed, 11 Nov 2009 09:17:57 -0800

Hi,

I'm going to do several FACS experiments using between 2 and 4 antibodies. My question may appear maive but I'm wondering if I should add each antibody to each sample separately or if I can make an antibody mix and then add the mix to each sample. Is there a risk that the antibodies bind to each other in the mix or not?
I also read a topic about not using trypsin to detach cell in culture when using Ab against membrane receptors? My targets are not receptors but are protein membrane so I guess I shouldn't use trypsin neither?
Thanks in advance for any advice

how to get sharp band for restriction enzyme

Wed, 11 Nov 2009 09:02:20 -0800

hi admin and chatter,

i would like to ask about, how to get sharp band becoz i get my restriction enzyme was not a sharp band, for your information i already use thin, thinnest well and also increase agarose gel into 2% until 3%. but still not get a sharp band.

i using 6x loading dye. my REs is mnII, hpy, cky,dde1 and total 8 re.
it is becoz of my loading dye?

thank you.

MLST of Candida glabrata

Wed, 11 Nov 2009 07:47:34 -0800

Im trying to type my C. glabrata isolates using MLST ..... Can anyone please quote a reference that gives the exact amplicon size of the six housekeeping genes FKS, LEU2, NMT1, TRP1, UGP1 and URA3 that we amplify and subsequently sequence ....

The references that i have gives information about the sequenced fragment size alone

asymmetric PCR

Wed, 11 Nov 2009 07:03:35 -0800

Hi there!
I've heard that I can carry out an asymmetric PCR in order to have a ssDNA, is it right? And then, could anybody suggest a protocol to carry it out?

Thanks!

promoter of a gene

Wed, 11 Nov 2009 07:01:02 -0800

Hi everybody!
I need the sequence of a gene promoter. I'd like to know how I can deduce it from the gene Bank.
Thanks!

Floating cells...pls help

Wed, 11 Nov 2009 06:37:36 -0800

Are all floating cells in a cell culture dish dead? will the floating cells get adhered to the dish surface after sometime?

Best time to isolate RNA for qPCR?

Wed, 11 Nov 2009 05:43:59 -0800

Hello everybody,

I have the following question, maybe a bit naive: If you add certain substances to a monolayer of cells and you want to check if these substances affect the expression of a gene, when would be the best time after adding the substance to isolate the RNA from the cells for qPCR. Obviously, it is very dependent on the gene, but in your experience with the genes you have investigated - how fast is the gene expression altered by adding e.g. growth factors to the cell culture media and how long is the gene expression kept in upregulated/downregulated state by these molecules? To be more specific, I am interested in investigating the expression of certain genes involved in cell migration and I want to check if their expression is altered by adding different growth factors to the cells. I will obviously have to isolate RNA at different time points. But I was just thinking - whether it would be wise to start the RNA isolations from 1-2h or from much later timepoint - let's say 6-12h after adding the growth factors? What would you do?

Making ATP solution

Wed, 11 Nov 2009 04:36:40 -0800

Help...........
I am trying to make a 10mls of ATP 10Mm solution the MW is 605.2

My balance is in grams and I am struggling...
any help would be good.

Transcription factor

Wed, 11 Nov 2009 01:10:51 -0800

Suppose I have (what I hypothesize) is a transcription factor. I want to find out if this binds to a sequence specific DNA, and if so what the sequence is. Next I want to find the genes that are turned on by it. Can you help me about how to go with it. A plan?

Fixing tissue

Tue, 10 Nov 2009 21:11:12 -0800

Has anyone tried immunohistochemistry on non-fixed tissue samples????
also which fixative in your experience is the best to be used for IHC????

simple pipetting problem

Tue, 10 Nov 2009 20:27:11 -0800

I feel kind of ridiculous posting this, but when I try pipetting Sepharose beads after washing and spinning them...well, I can't. They don't pipette up. I've cut the tips. Do I leave some of the wash buffer in there or what? I'm just using these beads initially to cut down on nonspecific binding. I don't have any problems when I need to pipette PGS beads. Suggestions? (Sorry if this seems pretty elementary..)

Storage of overexpressed protein

Tue, 10 Nov 2009 18:08:31 -0800

Hi everybody,

Im working with protein purification and for that purpose ive stored my overexpressed protein (E. coli BL21, host) as cell pellets in -20 freezer for about 4 weeks. Is there any problem in storing overexpressed protein like this. or is storage of overexpressed protein in -70 most essential ?
pls help
thanks

tRNA

Tue, 10 Nov 2009 14:30:17 -0800

Hi,

A maybe dumb question. For the hybridisation buffer tRNA (torula yeast RNA) is one of the components and I've bought powdered tRNA from Sigma.... How do I make it up? I've found web pages that say that I should digest it with proteinase K and extract using phenol chlorofom before I can use it. Is this necessary?

Isolation of membrane proteins from Red Blood Cells

Tue, 10 Nov 2009 14:16:34 -0800

Hi to all,

Does anybody have a protocol to isolate proteins from red blood cell? I tried an Erythrocyte Fractionation protocol (http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab1.html) however it was messy and the results were unreliable. I tried again with heparin tubes to avoid red cell agglutination, but I still did not get reliable protein extracts. Any suggestions or guidance would be greatly appreciated!

PCR amplifying 50bp ssDNA ?

Tue, 10 Nov 2009 13:19:23 -0800

Hi,

I need to amplify small 50bp ssDNA using PCR. How to design primers fitting in this small 50bp region ? Any ideas .. Highly appreciate it.

Thanks,

kmad