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How to normalize my samples before I transfect my cells


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#1 new2science

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Posted 02 May 2010 - 09:11 AM

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.

#2 pDNA

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Posted 02 May 2010 - 09:23 AM

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

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

Regards,
p

#3 new2science

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Posted 02 May 2010 - 09:27 AM

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

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

Regards,
p


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?

#4 pDNA

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Posted 02 May 2010 - 11:27 AM

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

#5 new2science

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Posted 02 May 2010 - 01:24 PM

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


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?

#6 pDNA

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Posted 02 May 2010 - 09:06 PM

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/Ál ...this is for sure genomic DNA or some other impurity.

Regards,
p

#7 new2science

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Posted 03 May 2010 - 03:55 AM

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/Ál ...this is for sure genomic DNA or some other impurity.

Regards,
p


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.




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