8 replies to this topic

[susanna]

Hi,

I would like to concentrate my DNA sample.
At this moment, i'm trying to maximize my DNA sample during extraction, by using low elution volumes and a higher amount of input material.
By doing this my concentration enhanced from 68 ng/ul to 156 ng/ul.
Is there an additional solution to enhance my DNA concentration besides lower elution volumes?

cheers,

Susan

[Thapa]

1. Warming elution buffer (upto 55 C) may greatly enhance the yeild sometimes.
2. Holding elution buffer 1-2 minutes before centrifugation (if this step required like in miniprep) is also recommended.

[Astilius]

You could use Amicon units. They are supplied by Millipore:
http://www.millipore.../item/ufc503096
http://www.millipore.../item/ufc5100bk

These have 30kdalton and 100kDalton cuttoffs but they also do other cuttoffs depending on how long your DNA is that you want to retain.
Here's the brochure:
http://www.millipore...2575b7005e90a6/


I used to use their predecessor, the Microcon, to great effect and I don't suppose the Amicon is any less capable. They're not inexpensive but they can concentrate DNA well and also help get rid of salts and other contaminants.

[susanna]

Thanks, i'll try it out

[rmbio]

You can also try larger elution volume first to increase the DNA yield and then can (1) precepitate the DNA with ethanol/isopropanol or (2) dry the whole eluent in chilled vaccume evaporator (eg. SpeedVac) and dissolve the pellete/dried sample in the minimum volume of water/TE to have higher concentration

[susanna]

rmbio,
so after eluating DNA, i just have to add ethanol/isopropanol to the eluaat? How many?
Do you have a protocol for this?
thanks!

[rmbio]

Protocol is pretty easy to follow...
1. Add equal volume of chilled isopropanol, mix and incubate at -20 C for 30 min
2. Pellet down the DNA by centrifuging at 10000 g / 10 min / 4 C
3. If you have used elution buffer (usually TE) for elution, give 70% ethanol was to the pellet (add 1 ml 70% ethanol, incubate at RT for 5 min, spin 10000 g / 10 min / RT). If you have used water for elution, no need to give 70% ethanol wash
4. Dry the pellet (after complete removal of the supernetant using pipette) at RT for 10 min
5. dissolve in the desired volume of water/buffer

[susanna]

Thanks

[mdfenko]

don't forget to add salt (the 70% ethanol wash is to remove residual salt after pelleting).

Edited by mdfenko, 11 July 2012 - 06:05 AM.