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DNA concentration...

by karthik3k
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karthik3k
#1
Jan13-05, 02:32 AM
P: 151
Is there by any way one can concentrate the DNA in a solution ???
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iansmith
#2
Jan13-05, 06:13 AM
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You can measure it either using a UV spectrometer or by gel electrophoresis.

With a UV spectrometer:
First diluted you DNA (I usually use 5 uL in 995 uL). Then adjust the spectrometer at 260 nm with your blank. Measure your DNA sample. You can then use the following formula to estimate the DNA concentration:

concentration = 260 reading * Absorbance factor * dilution factor (in my it is 200)

Absorbance Factor
ssDNA: 37 g/ml
dsDNA: 50 g/ml
ssRNA: 40 g/ml

You can also measure the purity sample by calculating the ratio of the A260/A280

Using gel electorphoresis,

run your sample with a molecular marker that has stantard DNA concentration. You then take the picture. Depending on the imaging material you will have to use different things. If you have a imaging hardware and software, there is a program that allows you to measure the intensity of a marker band of a size identical to your DNA sample and you can compare it to the intensity of your DNA sample. You then use the concentration of the stantard to estimate the concentration of your sample.
karthik3k
#3
Jan13-05, 06:18 AM
P: 151
thanx ian,
but my actual question was to concentrate, i mean increase the concentration of DNA in a solution.

or to decrease the volume of the sample without decreasing the amount of DNA present in it...

iansmith
#4
Jan13-05, 06:22 AM
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DNA concentration...

I should be on PF a few minutes after I wake up or before breakfeast.

You can precipitated your DNA with cold 95% EtOH. Collect the DNA by centrifugation (14 000 g for 10 minutes) and resuspend in a smaller amount of water or buffer. Yield tend to be around 80%. The colder the EtOH, the greater the yield. You cold aslo put your sample on ice for about 10 minutes prior to centrifugation to increase the yield.

There is also columns to concentrated DNA concetration and the should be between 80% to 95% depending on the columns.

You will of course lose some DNA but your concentration should increase.
karthik3k
#5
Jan13-05, 06:26 AM
P: 151
i have tried the column stuff..(gel elution kit), but the efficiency is very low, n the amount of DNA to be eluted is ~ 500ng.

neways i never tried ethanol stuff....
Do u have any protocol / paper regarding that ???

thanx
iansmith
#6
Jan13-05, 06:32 AM
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Your yield will be low if you put too much DNA in your column. The column have a maximun holding capacity as described in this protocol
http://www.proteigene.fr/pdf/d4005inst6-7-02.pdf

This will influence your yield.

For EtOH precipitation, use 30 uL of 95% EtOH per 10 uL of sample. The final concentration of EtOH should be around 70%. You add the cold EtOH, put on ice for 10 minutes, centrifuge for 10 minutes at 14 000 g and then resuspend in water or buffer.
karthik3k
#7
Jan13-05, 06:38 AM
P: 151
oh ok thanx :)
DocToxyn
#8
Jan13-05, 07:16 AM
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The method for nucleic acid precipitation in Current Protocols in Molecular Biology calls for the addition of monovalent cations to the mix prior to ethanolic precip. Check your library for a copy of this very handy reference source.

Four different cations are suggested, depending on characteritics of the DNA/RNA solution. Most common is sodium acetate- stock solution of 3.0 M (pH 5.2) added to a final conc. of 0.3 M. Then 2 volumes of ice-cold ethanol, mix well, sit for 15-30 min on ice and spin. Other cations include ammonium acetate final conc. of 2.0-2.5 M, lithium chloride final of 0.8 M and sodium chloride final of 0.2 M (use this if SDS is involved). I've used this and similar protocols for isolation and concentration of genomic DNA (tail snips), plasmids, in vitro transcribed RNA probes, etc.
iansmith
#9
Jan13-05, 07:55 AM
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DocToxyn made me realized that we have Potassium Acetate (2.5%) in the 95% ethanol solution.
Monique
#10
Jan13-05, 09:33 AM
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The quick (and dirty) method is to evaporate water out of your sample by using a speedvac centrifuge. I found Qiagen columns to work really well, but they're expensive.
karthik3k
#11
Jan13-05, 11:01 AM
P: 151
????
wont u change the concentration of the salts other stuff in the solution if u evaporate ????

i have used QIAgen kits for elution, but i feel it to be less efficient ....
Monique
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Jan13-05, 11:16 AM
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yes, you will: that's why it's called a dirty method
Moonbear
#13
Jan13-05, 09:28 PM
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Adding salts will help with the precipitation, but then your sample has salts in it. You'd then need to add a step to clean it to get a more pure sample if the salts will interfere with what you're doing with the DNA. I do this too infrequently to remember a protocol off the top of my head (and I'm still in the lab doing something else, so don't have time to go hunting for a protocol right now). I'm sure Monique or Ian do this far more often to have one at hand.
rockind78
#14
Jan14-05, 11:54 AM
P: 78
http://arbl.cvmbs.colostate.edu/hboo...anip/conc.html

I like isopropanol instead of ethanol, but that is more of a personal preference thing. Good luck.
Sankt
#15
Dec27-10, 01:47 PM
P: 1
Besides UV spectrometer, is there any process to determine DNA concentration?Can Computer simulation be used?


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