Hi,

1) 1-5 freeze thaws would not effect DNA integrity much. Recheck the plasmid concentration. Use the plasmid after whole tube get thawed and mixed thoroughly. There is a chance for precipitation of DNA.

2) Transfection efficiency should be considered. (There are some hard to transfect cell lines. / Transfection reagent might be problem.) So, try to include EGFP plasmid( equal to 1/10th of your plasmid concentration) to make sure the procedure is flawless.

If you store your prep'd DNA (assuming this has been done correctly and the sample is clean) in a) TE buffer, and b) autoclaved/sterile/DNase free plastics there really shouldn't be any issue of degradation of your sample, I've had plasmids that I've stored at 4 deg C for over a year without a drop in transfection efficiency though I always keep aliquoted stocks at -80 deg C.

If samples are stored in water only they become acidified (CO2 dissolving in unbuffered solvent) which can leave the DNA susceptable to hydrolysis.

I agree with the comments given above. Most probably, your plasmids might have been degraded. You can repeat the experiment with freshly prepared plasmids.

Thanks for your answers.

All of my successful constructs have been prepared with the same commercial miniprep kit and eluted with the same elution buffer and stored at the same freezer. I always thaw completely, mix and spin down before using.

But it only happens to "some" genes, while expression of other genes cloned into the same backbone vector (the same restriction enzyme sites) are normal (although the latter ones have been stored/freeze-thawed multiple times for the last several years).

First thing check integrity of plasmid DNA on agarose against those plasmids that perform well, re-check concentration. You may well have nuclease activity present

It certainly sounds like plasmid  integrity is lost in these preps. Despite the fact you use the same kit to purify all plasmids it is not a fool proof way of obtaining clean plasmid.

Make a new prep and repeat tfx using other plasmids that performed well as positive controls

It happens. Just do a transformation and isolate plasmid and do transfection.  This should work.

make a glycerol stock always, they never go bad

Before doing transfection you can check on the gel and it will help you.

It could be because of DNA degradation and this can be avoided by using filter sterilized TE buffer and at the same time aliquot the plasmid as 50ul each and store at -20C. 

Hi Hong-Su,

I have to agree with most of the considerations above. Independently from how you prepare your plasmid DNA, it never comes out with the same purity. I'm not sure what kits you are using to prepare the DNA, but keep in mind that if you use kits with any type of positively charged beads (not membranes) they often disaggregate and microparticles come down with the DNA, although you can't see them. They strongly inhibit enzymatic reactions on the DNA and transfection efficiency. Either you spin your resuspended DNA at the ned of the prep for 10 min at max speed and transfer the super into a new tube leaving 40-50 ul at the bottom of the tube (sometimes you'll even be able to see the tiny pellet from the material) or use kits that exploit properties of membranes.

In any case, it would be good to follow Ian's suggestion and check your plasmids that give good yield compared to those that don't on an agarose gel, loading equal amounts (at least theoretical...). You might be surprised...

Good luck.

Hi,

Above suggestions are good. You should also check your micro centrifuge tube quality. it should have low DNA affinity.