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Transfection

Transfection - DNA Techniques

Transfection - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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Old 03-26-2013, 12:11 AM
Pipette Filler
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Arrow Transfection



Hello all!

I am currently using the FuGene system to transfect HeLa cells with DNA that I have measured using a nanodrop machine.

The nanodrop gave a reading of 400ng/ul of plamid DNA from my extraction.

The FuGene system requires that I do a 3ul Fugene:1ug DNA ratio.

I am a little unsure as to how I should be calculating the volume required of DNA to achieve this ratio.

I know that 1ug=1000 ng/ul, therefore I have divided 400ng/ul by 1000 to give 0.4 ug/ul. Now I am stuck.

This is probably simple but I'm new at this! Thank you.
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Old 03-27-2013, 10:58 PM
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Default Re: Transfection

Any help would be greatly appreciated
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Old 04-26-2013, 09:56 PM
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Default Re: Transfection

kgirl,

A month late, but hopefully I can help. The volume and amount of DNA you use is entirely dependent on how many cells you have and how efficiently you want the transfection to go. But anyway you're right that right now you have your plasmid at a 0.4ug/ul and you know that you you should use 3ul Fugene:1ug of DNA. So, for example, if you used 1ul of your DNA (0.4ug) by simple ratio you should use 1.2ul of Fugene reagent:

1ug DNA/3ul Fugene = 0.4ug DNA/xul Fugene (x=1.2)

You can substitute the amount of DNA you actually want to use where I put 0.4ug and solve for x to get volume of Fugene for that amount. I used to do this on 70-80% confluent cells in 96 well plates and use 0.1ug DNA + 0.3ul Fugene6 reagent per well (my DNA was a more dilute than yours so it came out to about 5ul per well). Good luck!
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calculation , plasmid dna , transfection


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