Low transfection efficiency using PEI - I'm trying to transfect BHK-21, with a large size plasmid (13kb), (Sep/06/2007 )

I'm trying to transfect BHK-21, with a large size plasmid (13kb), but getting extremely low transfection efficiency, 1-5%. Can anyone help me in this regard. I'm using 25kd, linear PEI. Can anyone suggest me methods for improving transfection efficieny of large size plasmids? please help

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Can you tell us the details of transfection experiment? Have you done transfection with 5-7 kb plasmid with LPEI before? What was the transfection rate with that?

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No, i din't tried LPEI for 5-7kb plasmids. I'm using 1ug DNA (13kb, diluted in optimem)with 2, 3, 4 AND 6ug LPEI(diluted in optimem). mixing both by vortexing, keeping for 10min for comlex formation and then add G.M.,and transfer to 24 well plates.

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Too much LPEI. Did you notice any serious toxicity?

Try to use 1:8 to 1:20 charge ratio (of PO4 to amine) using DNA monomer MW=330 and LPEI monomer MW=43 to calculate. You dont need more than 1:2 weight ratio for DNA to LPEI. You can transfect cells in the presence of up to 5% FBS.

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oh yes, I observed too much cell death and very slow growth rates of surviving cells. To get N/P raitio of 1:8, with 1ug DNA exactly how much LPEI,25kd, should be used. Can u plz help me do the calculations, i'm not getting it. plz help

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1ug DNA has ~3 nmoles PO4 (1000 ng/330 ng per nmole), 3x8=24 nmoles LPEI needed.

24nmoleX43 ng/nmole=1032 ng will be the amount of LPEI to use for 1:8 ratio.

Ratio between 1:8 to 1:15 would be enough. 1: 20 if you want to see the full range.

I think you can use up to 1.5 ug DNA per well for 24 well size, but do the calculation again to use the correct amount of LPEI.

Use cells that are 80% confluent.

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THANKS A LOT.!!!,
Well will u plz tell me what other reagants can b used to get high transfection efficiency for large size plasmids?

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13 kd plasmid is OK for Lipofectamine 2000 as well. Its just you have less copys of plasmid delivered per ug than 5-7 kd plasmid. Where did get LPEI from? was that expensive? Lipo2000 is pretty expensive. It is better than LPEI.

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I used, Sigma aldrich LPEI, available in my lab. I also tried with superfact(Qiagen), but same results.(abt 10% transfection) I'll try to purchase Lipofectamine 2000.
Thank! u really helped me a lot, i'm greatful for that!

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FuGene 6 from Roche is supposed to be the best transfection reagent for plasmids... I personally use Lipofectamine2000 for oligonucleotide transfections and it's good.

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