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lipofectamine and PEI transfection of hela cells


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#1 jasmine0507

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Posted 25 February 2010 - 11:41 AM

HI All,

1.I want to transfect HeLa Cells with lipofectamine plus reagent to optimise the quantity of DNA and lipofectamine to be used to get the best results.
I will be using 24-well plate for transfection.

I would like to use lipid/DNA ratio for the optimisation. Could any one please explain me how to calculate this ratio and what should be ratios
I can use to get near the best possible results.

2.Similarly, for jetPEI and 25kDa Branched PEI, how can we calculate the N/P ratio.

3. Is there any simple method to check the cytotoxic effects of these reagents after transfection ?

Thank you.email :

#2 laurequillo

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Posted 25 February 2010 - 01:40 PM

HI All,

1.I want to transfect HeLa Cells with lipofectamine plus reagent to optimise the quantity of DNA and lipofectamine to be used to get the best results.
I will be using 24-well plate for transfection.

I would like to use lipid/DNA ratio for the optimisation. Could any one please explain me how to calculate this ratio and what should be ratios
I can use to get near the best possible results.

2.Similarly, for jetPEI and 25kDa Branched PEI, how can we calculate the N/P ratio.

3. Is there any simple method to check the cytotoxic effects of these reagents after transfection ?

Thank you.email :


Normally in the datashet they tell you how to check that ratio. Normally is like "2-3ul lipid per 1ugr of DNA " something like that. You have to check but if i recall was something like that.

If you want to check the ratio you can just make a titration :1/1, 1/2, 1/3, 1/4, 1/5 using for example GFP and then check the cells in the microscope, or jus t checkin by wb.
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#3 jasmine0507

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Posted 26 February 2010 - 12:35 AM

Normally in the datashet they tell you how to check that ratio. Normally is like "2-3ul lipid per 1ugr of DNA " something like that. You have to check but if i recall was something like that.

If you want to check the ratio you can just make a titration :1/1, 1/2, 1/3, 1/4, 1/5 using for example GFP and then check the cells in the microscope, or jus t checkin by wb.
[/quote]

Actually, what I am doing is the part of practical work in my masters program. We had been givena list of things available tp us and we have to design our own protocol.So , we decided to transfect hela cells using 3 diff reagents in order to compare their tranfection efficiency and cytotoxic effects. We dont have to use any standard protocols, all we can do is take an idea from them. Now to compare the three techniques, we want that each well in the 24-well plate should have the same no. of cells and same quantiy/conc of reagents and may be just vary quantity of DNA to vary the ratios : N/P and lipid/DNA.
In order to plot a graph later,I would like to work on same ratios for the three reagents. So I need an idea, what would be those ratios, around which the three can give results. I found some publications, doing exactly what i want, but they are paid, so i am unbale to see them.

Moreover, I have some slight idea. We need to calculate so equivalents,for pei, equiv= charged nitrogens in PEI/charged phosphate in DNA.
I also know that we might need some intial conc. like data to calculate this. I have another info :

0.28 ul PEI 100mM/ug DNA = 9 equivalents.




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