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.
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How to normalize my samples before I transfect my cells
Started by new2science, May 02 2010 09:11 AM
6 replies to this topic
#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
or can you give us a more detailed description on your problem.
Regards,
p
#3
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Posted 02 May 2010 - 09:27 AM
pDNA, on May 2 2010, 12:23 PM, said:
either dilute or concentrate? ...how about that?
or can you give us a more detailed description on your problem.
Regards,
p
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
...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
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Posted 02 May 2010 - 01:24 PM
pDNA, on May 2 2010, 02:27 PM, said:
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
...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/µl ...this is for sure genomic DNA or some other impurity.
Regards,
p
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
#7
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Posted 03 May 2010 - 03:55 AM
pDNA, on May 3 2010, 12:06 AM, said:
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
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
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.
