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Thread: GFP- Green-Flourscense-Protein Plasmid

  1. #1 GFP- Green-Flourscense-Protein Plasmid 
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    Hi there!

    I got some questions that I hope somebody has the answers to.

    Im currently using an transfection method called electroporation. And the idea is to electroporate hippocampal slices from rats using a plasmid containing GFP. It seems that the transfection method works however there are some problems. I tried to see if the plasmid could somehow transfect the cells without any use of the electroporator. I added 31 ul onto the slices and by looking at the microscope i actually can see green light. And by comparing it to a positive control it looked quite similiar.
    So my question is can the plasmid somehow been taken up by the cells spontanseouse? Is this possible? But it seems so wierd. I must add that I exposed the slices with an plsmid for 1 day, and then looked at the microscope.

    Cheers
    Kristoffer.


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  3. #2  
    Forum Junior AndresKiani's Avatar
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    Quote Originally Posted by neverion View Post
    Hi there!

    I got some questions that I hope somebody has the answers to.

    Im currently using an transfection method called electroporation. And the idea is to electroporate hippocampal slices from rats using a plasmid containing GFP. It seems that the transfection method works however there are some problems. I tried to see if the plasmid could somehow transfect the cells without any use of the electroporator. I added 31 ul onto the slices and by looking at the microscope i actually can see green light. And by comparing it to a positive control it looked quite similiar.
    So my question is can the plasmid somehow been taken up by the cells spontanseouse? Is this possible? But it seems so wierd. I must add that I exposed the slices with an plsmid for 1 day, and then looked at the microscope.

    Cheers
    Kristoffer.
    At what temperature were the samples kept at? Do you see a coat, colony, small specks? Temperature and lighting may allow some of those cells to be successfully transformed.


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  4. #3  
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    Hey!
    Sorry for the late reply. I put the samples in an incubator (37 degree C with 5 % C02)
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  5. #4  
    Making antisense Jon Moulton's Avatar
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    Be sure to compare with cells that have not been exposed to the plasmid. Some cultured cells naturally fluoresce in the green band (autofluorescence).
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  6. #5  
    Forum Professor Zwolver's Avatar
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    Quote Originally Posted by neverion View Post
    Hi there!

    I got some questions that I hope somebody has the answers to.

    Im currently using an transfection method called electroporation. And the idea is to electroporate hippocampal slices from rats using a plasmid containing GFP. It seems that the transfection method works however there are some problems. I tried to see if the plasmid could somehow transfect the cells without any use of the electroporator. I added 31 ul onto the slices and by looking at the microscope i actually can see green light. And by comparing it to a positive control it looked quite similiar.
    So my question is can the plasmid somehow been taken up by the cells spontanseouse? Is this possible? But it seems so wierd. I must add that I exposed the slices with an plsmid for 1 day, and then looked at the microscope.

    Cheers
    Kristoffer.
    For short, yes. A cell can spontainously accept plasmids. Due to radiation (light, gamma, alpha or beta), due to a slight temperature difference, due to a slight pH potential, due to mechanical influences (simply pushed in), an osmotic imbalance, in a restructuring or repairing of the cell wall/membrane. So you see, plenty of possibilities.

    I however believe that flushing the sample is your main problem, a lot can happen in 1 day. Use a better transfection method. Like a phage, a simple heat shock, or an enzyme linked transfection.
    Growing up, i marveled at star-trek's science, and ignored the perfect society. Now, i try to ignore their science, and marvel at the society.

    Imagine, being able to create matter out of thin air, and not coming up with using drones for boarding hostile ships. Or using drones to defend your own ship. Heck, using drones to block energy attacks, counterattack or for surveillance. Unless, of course, they are nano-machines in your blood, which is a billion times more complex..
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  7. #6  
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    Hey!

    I really wished that i could attach some pictures here, but it seems that I cant, because of the error that accours.

    However, I think that spontanouse uptake of the plasmide have accoured. I even tried to compare organotypic slices which were Non-exposed to organotypic hippocampal slices that have been exposed for a plasmide for 1 day and also exposed for 2 days and a significant difference is actually seen. You clearly can see the cells in the green fluorescence. But also that the cells that were exposed for 2 days show a more bright and "finer" cells.
    But my thought were that damaged cells somehow could have taken up the plasmide and therefore you see a bright green color, and therefore I tried to wash the cells with PBS 3x5min, but after have spoken to my supervisiour it seems that damage cells cant take up the plasmide due the machinery that it needed for e.g an functional ER or Golgi. This would therefore indicate that it is healthy cells that takes up this plasmide.


    But this actually leads to another question. And this is about the microscope. When using 4x 300ms, 10x300 and 20x200ms it seems that all of the slices even the one that actually havent been exposed to plasmide will lighten up and a green colour is seen.Though, the c But if you compare this to 4x200ms 10x100 and 20x20 zoom, the ones that havent been exposed to a plasmide was dark. So it seems that the microscope could also give rise to a false-positive. Have you someone come across of this? Would this indicate that to much "backround light exposure time" could lighten up the cells as well?

    Anyways. Im sorry for any misspells or if you guys have a hard time to understand what Im trying to say.

    Have a nice day
    Cheers
    Kristoffer.
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  8. #7  
    Making antisense Jon Moulton's Avatar
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    If you allow the machine to collect enough photons, you see autofluorescence from untreated samples. Autofluorescence in green is pretty common.
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