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Hek293 transfection help!!!!

Hek293 transfection lipofectamine 2000 DNA plasmid calculations

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#1 Jasleen



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Posted 13 July 2015 - 09:20 AM

I need to transfect HEK293 cells with myc-tagged rat kappi opioid receptor (myc-rkopr) using lipofectamine 2000.
Protocol says
1) in eppendorf add 3ul lipofectamine 2000 and dilute with Optimem to 50ul.
2) in another eppendorf add DNA plasmid and dilute with Optimem to 50ul so its concentration is 0.7ug/ml.
3) incubate both tubes for 5min at room temperature
4) mix content of both tubes together and incubate for 20min
5) add 100ul of the mix to a 35mm plate of HEK293 seeded at 1.0x106 cells/ml.
6) incubate for 18-48hr at 37 °C.

So my myc-rmyc-rkopr has a concentration of 734.1 ng/ul. How much DNA and Optimem do I add so that the final DNA concentration is 0.7 ug/ml?

#2 bob1


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Posted 13 July 2015 - 01:14 PM

so you already have it at approx 0.7 ug/ul... if you took 1 ul and put it into 1 ml you would have 0.7 ug/ml


What fraction of 1 ml is 50 ul? Note to get a usable volume, you are going to have to dilute the DNA anyway.


Note that in your protocol it says to add lipofectamine then dilute with optimem. Always put the optimem in the tube first then add the lipofectamine, as these lipid based complexes don't like interacting with plastic much.

Also tagged with one or more of these keywords: Hek293, transfection, lipofectamine 2000, DNA plasmid, calculations

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