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Zee's Content

There have been 8 items by Zee (Search limited from 15-April 20)

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#157060 NanoDrop UV-Vis spectrophotometers

Posted by Zee on 28 June 2013 - 05:12 AM in General Lab Techniques

I've read the protein concentration results given by Nanodrop are not reproducible nor accurate due to variability in the types of proteins present in different samples. I tried it once and compared results to concentration values from a cuvette-based spectrophotometer, and the results were inconsistent with no obvious pattern. That being said, I only looked at 2 samples that one time. If someone has gotten the nanodrop to work for measuring protein, that would be awesome!

#157059 HEK293 vs. SH-SY5Y Cell lines? Which best for in vitro Alzheimer's Model?

Posted by Zee on 28 June 2013 - 05:03 AM in Tissue and Cell Culture

Have you considered testing both lines? I always like it when data is replicated in 2 different kinds of cell lines in publications. Personally, I would start with HEK293 and then confirm the results in SH-SY5Y if it looks promising. This is because HEK293 is easy to obtain, grow, and manipulate for experiments.

In different labs, I've grown HEK293 in DMEM+10% FBS (heat-inactivated)+1%Pen/Strep and in DMEM/F12 50/50+10% FBS (heat-inactivated)+1%Pen/Strep. The cells grew faster in the DMEM/F12, but that could have been a stock difference.

Note: The origin of HEK293 is a topic of debate. It expresses many neuronal markers and has been hypothesized that it originates from a neuronal lineage rather than the kidney tissue from which it was isolated (Shaw,G., et al, 2002). While historically some labs have used it as a negative control and compared it to neuronal lines, it is not the best model. That being said, I think it makes a great tool for neurobiologists & researchers looking at AD.

#157057 Sybr Green vs Taqman -- a practical approach

Posted by Zee on 28 June 2013 - 04:44 AM in PCR, RT-PCR and Real-Time PCR

My PI wants me to switch from using Taqman to Sybr green to look at expression of a non-endogenous gene. Mainly the switch is to safe money. I'm not convinced that this is the best idea as Taqman is supposed to be more accurate. Also, a friend of mine said that her old PI determined that Sybr was not that much cheaper. She said this was because of the large number of controls you have to run. Since I have never run a Sybr green experiment so am unfamiliar with the number of controls required, I was wondering if anyone has compared the costs between the 2 methods. How many controls are you supposed to run? I know when you get a new primer set you have to run a standard curve, but you don't have to do that every time, do you? Are there additional controls I am not thinking about?

If you have any arguments for either method please let me know so I can either agree that the switch is a good idea or approach my PI with arguments to not switch.

Thanks for your help!

#157055 What type of Northern Blot should be used for smaller transcripts?

Posted by Zee on 28 June 2013 - 04:35 AM in General Lab Techniques

Thank you!

#156925 Less than 10 GFP positive cells

Posted by Zee on 25 June 2013 - 11:18 AM in Flow Cytometry

Yes, this is commonly done. Flow cytometers are more sensitive to GFP signal than the naked eye so they can detect GFP positive cells when you do not see them in the fluorescence microscope. For certain experiments on cells with low transfection efficiencies, I will sort out the GFP positive cells by FACS and use those in my next assay.

just make sure you have a negative control, no GFP expressing cells. If your wanting to look at cells from transgenic mice, get a sample from a mouse that is not supposed to express GFP. If this is from transfected cells, set aside a aliquot of cells that were not transfected. These controls are vital to determining from where your GFP signal is coming. All cells have some level of background fluorescence and this differs between cell lines and cell types... so make sure the cell types are the same.

#156924 FACS using DAPI for apoptosis analysis

Posted by Zee on 25 June 2013 - 11:11 AM in Flow Cytometry

I don't know anything about using DAPI by flow. I wonder if it is detectable?

However, there are additional alternatives. Try Hoescht 33342 from invitrogen. It fluoresces in the UV range, which I assume DAPI will too, but I know it works by flow. You do have to check that the equipment you are using has the correct filters and laser, though.

#156923 PI cell cycle analysis and apoptosis - or would a different stain be better?

Posted by Zee on 25 June 2013 - 11:07 AM in Flow Cytometry

PI is a good standard for looking at cell cycle. However, if you are wanting to look at specific stages within the cell cycle, it is considered a crude assay. For example, if you want to look at apoptosis, Annexin V is typically used. If you want to look at proliferation, I would recommend performing an EdU staining (equivalent to a BrDU assay but a little simpler to do in lab). For both of these experiments, PI analysis is done in conjunction with the additional stainings, either for Annexin V or EdU, that tell you more about what the cells are doing. Most companies have kits and detailed protocols for both of these, including invitrogen and eBiosciences. (I usually recommend using invitrogen's SOPs while the reagents are cheaper from eBiosciences.)

#156922 What type of Northern Blot should be used for smaller transcripts?

Posted by Zee on 25 June 2013 - 10:58 AM in General Lab Techniques

I am trying to figure out how to look at RNA transcripts which are between 200-500bp in length. Most protocols are either for full length transcripts, 1-2kbp, or miRNA, ~20bp, and the protocols vary dramatically for these 2 types. Is there a special type of gel I should use for looking at a transcript between these 2 sizes? I was thinking about simply increasing the concentration of my agarose gel and follow the protocol for full lengths transcripts. Anyone have any ideas about whether or not this will work?

Thank you for your help!

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