How to normalize my samples before I transfect my cells - (May/02/2010 )

Hi,

I was told that before I perform my transfections that I should be normalizing my plasmid DNA samples to 1ug/ul. I don't know how to do this. I did my purifications of my plasmids using QIAGEN maxi preps and I found out the concentrations of each sample using a nanodrop but now I don't know how to get each of my samples to be 1ug/ul? How does one go about normalizing samples?

Thanks.

either dilute or concentrate? ...how about that?

or can you give us a more detailed description on your problem.

Regards,
p

pDNA on May 2 2010, 12:23 PM said:

Some of my samples are really low. For instance:

sample 1: 4.96 ng/ul

sample 2: 57.14 ng/ul

sample 3: 540.44 ng/ul

sample 4: 144.58 ng/ul

How would I concentrate this?

evaporate the solvent?

...normally you do this by using a vaccuum concentrator.

for maxipreps it is really poor yield ...what is your volume of your sample?

best regards,
p

pDNA on May 2 2010, 02:27 PM said:

I resuspended the pellet in 50 ul of sterile double distilled water (I did this because the pellet I got was very small). Even with the mini preps I was getting such low yields, so that is why I swithced to maxipreps. Do you think there is something wrong with my plasmid?

since you provided no information on your plasmid its hard to say if there is something wrong!

Is it a low or a high copy plasmid?

For a high copy number plasmid your yields are very low!

Maybe there is something wrong with your plasmid or with your protocol ...have you checked your plasmids on an agarose gel? I would not expect plasmid DNA from 1 liter broth at a concentration of 4 ng/µl ...this is for sure genomic DNA or some other impurity.

Regards,
p

pDNA on May 3 2010, 12:06 AM said:

Hi, I plan on running some 1% agarose gels today. I was told it was a high copy plasmid...I will double check that information today. Thank you for your help.