I am working with some DNA samples obtained with a phenol/chloroform extraction and I have many clumps that I can't resuspend in the buffer (TE). I tried heating for 10 min at 42C and with the tip of the pipette, but in some samples I don't manage to get an homogeneous suspension of my DNA. Does anyone have any tricks?
Paula,
The trick is to dry the DNA just enough so that the ethanol has evaporated completely but the DNA pellet is still moist (not completely dried up). I suggest that you get rid of the 70% ethanol and allow the DNA blob/pellet to stick to the side of the tube to allow the trace amounts of ethanol to pool at the bottom. This permits better evaporation. I usually put the tube in a 37 C incubator for 15-20 minutes or less to ensure all ethanol trace has vanished. Add 1X TE at pH 8.0 and leave in a 4 C incubator overnight and for a few days. The slightly alkaline pH allows good dissolution of DNA. Do not freeze/thaw DNA. There is a misconception that DNA can degrade if it is not frozen. Remember that DNA is one of the most stable molecules in nature. As long as you use TE or pure water, you should have no worries. DO NOT sonicate DNA. Sonication shears DNA to smaller fragments.....
But, don´t ultrasound break the DNA into too small pieces? I have no experience with water bath, to be fair
It very much depends on your protocol and what kind of DNA are you extracting?
Think about which contaminants could be in your prep and how you would remove them... ie proteinase or RNAase?
If the DNA is really pure you should maybe think of ways to relax it without destroying it but i guess that depends on what you need the DNA for.
Sometimes simply repeating the purification of your sample with fresh reagents will work.
Good luck.
Hi, Paula
It looks like your samples were not purified enough from proteins, so your contaminated DNA is in "insoluble form". You need to repeat your purification (with higher volumes or be more carefully with interphase).
Good luck!
The best way to re-suspend DNA without shearing it is keeping it at 37 degree water bath for 1-2 hrs. It does not have any adverse effect on the integrity of the DNA pellet.
Do not sonicate!! this will shear the DNA. If you have clumps it could be due to a carry over of protein, ethanol or because the you've got a lot of DNA and it is difficult to resuspend.. Is it genomic DNA or plasmid? I would try to add some more solvent and leave it overnight at 4ºC. If it is plasmid DNA you can also vortex the solution.
Estanis, it is genomic DNA.. Finally I got it solved by heating several times during 10 min at 65C and resuspending with the pipette after every heating. I hope I did´t damage the DNA with that process...
Paula,
The trick is to dry the DNA just enough so that the ethanol has evaporated completely but the DNA pellet is still moist (not completely dried up). I suggest that you get rid of the 70% ethanol and allow the DNA blob/pellet to stick to the side of the tube to allow the trace amounts of ethanol to pool at the bottom. This permits better evaporation. I usually put the tube in a 37 C incubator for 15-20 minutes or less to ensure all ethanol trace has vanished. Add 1X TE at pH 8.0 and leave in a 4 C incubator overnight and for a few days. The slightly alkaline pH allows good dissolution of DNA. Do not freeze/thaw DNA. There is a misconception that DNA can degrade if it is not frozen. Remember that DNA is one of the most stable molecules in nature. As long as you use TE or pure water, you should have no worries. DO NOT sonicate DNA. Sonication shears DNA to smaller fragments.....
TE buffer is good it helps to achieve complete solubilisation due to its slight alkaline nature. But water is not a buffered solution and hence it might lead to DNA degradation.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA