Hi, no you do not need to linearize your plasmid..unless required by your transfection method..which is? anyway once you transfect your plasmid, that MUST have an eucariotic resistance, you need to select for it...so how to do it...prepare some plates that you'll transfect without dna, then use a serial dilution of your antibiotic to determine which dosage kill your cell in 7- or 10-days..then use this dosage to select your transfected cells..after 7 or 10 days you''ll have some clone in the plate and you can pick it up (cloning ring) and you'll have your stable monoclonal cell line... hope this helps :)

Answers above sum it up, the general consensus is that linearized plasmids give better results but not necessary.

I do agree with the above answer by Krzystof Wicher that if you linearize your plasmid then chances of integration increases but the uptake of the plasmid is reduced. While if you have circular plasmid then it is vice versa. But if your plasmid is having selection marker then you can make a selection in the end and can get the best transfected.

No, it is not necessary to linearize a plasmid before making stable transfectants. We transfect without linearizing, and we get stable transfectants. However, maybe the number of clones we get would be higher if we tried linearizing; since we haven't tried, we don't know the effect on yield.

Dear Saurabh,

You have already received replies from experts using transfection. We have generated a number of stable inducible cell lines of both adherent and non-adherent nature and have used intact plasmid.

Ampicillin gene can be the optimal breaking point...

In mammalian cells this gene is not necessary...

Ampicillin and kanamicin resistance genes are the optimal point to cut, as Nicola said above. Usually there are unique restriction sites for ScaI an PvuI in AmpR and PvuI, NruI and SspI in KanR.

And again, in mammalian cells these genes are not necessary.

No, it is not necessary for stable transfection. the transfection efficiency is not important. Because the important one for stable transfection is to select expressing cells, it is better to do dose response first,

We directly compared linearizing vs supercoiled for stably transfecting 293Ts, U2OS, HT1080, and hamster lung epithelial cells. We found no difference and in some cases, linearizing the plasmid actually decreased our chances of obtaining positive clones in the end. This was with a 10.3 kb cDNA so the gene was very large. We actually spent a significant amount of time creating unique restriction sites to linearize this plasmid and in the end, it didn't make a difference. As long as you screen your clones in the end for full length expression, you will be fine.

293T is resistance to G418. I do not know about other drugs. 

As Ryan said it does not make sense to use linearizing plasmid. But we should consider if the plasmid are from mini prep or midi. I go for midi prep, because of less chance of toxicity of plasmid extraction reagents. 

 Guys I am using U2OS cell line with X-TREMEGene as a transfection reagent. 

a day after transfection I found lots of dead cells. I use 1:3 ration of reagent and DNA. my DNA is about 7 kb. Tranfecting with 2ug DNA and 6 ul of X-treme. is there any one who can help me. Thank you in advance 

How big is your construct? depends on that. Lower than 7kbp no need to linearize. Just first do a transient transfection. after 1-3 days replace the medium with selection antibiotics and continue for a week. Then trypsinize the reaming cells and move them to a 6 well plate or T25 flask and let them expand. you can make your 1st frozen badge after T25 becomes confluent. Call them polyclonal badge. Then depending on any other tag you have to make monoclonal transfected cell line.

Your transformets will have a range of expression of your gene, i would group them into high, medium, low, expressers, and none, toss these last ones, make freeser stocks of the others.

here is an article about the whole affair. Cutting does improve things, the key seems to be to cut in the right position. Cutting in the wrong position will have negative effects. As this was not tested systematically with many different plasmids, I´d just assume that cutting as far away from all important loci might be the best option. To cut right next to an important feature (promoter, UTR, polyA site, CDS...) is likely detrimental.

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