Hi, I wanna ask about DNA and RNA pellet.
I got the thin white spot at the bottom of the microtube. I assumed that this white spot "pellet" is a DNA pellet or a RNA pellet (ofc after I did DNA or RNA extraction).
but my friend just told me that, that was not a DNA or RNA pellet. it was just the residue from the samples and he said that the pellet of DNA or RNA is translucent. I didnt understand what he said, I looked at the bottom of the tube but nothing translucent "things" except the residue of Ethanol 70%. I tried to dry it to look after the translucent things but still nothing except the white pellet.
so what is the real pellet looks like?
*as far as my mentor told me that the white pellet after the extraction is the DNA or RNA pellet
the white things are usually proteins and other stuff from the degraded cell but they come out in an earlier phase of the procedure after a centufigation....if u took carefully the upper liquid, then treated with isopropanol as the protocol says and then centrufigation, then the thing at the bottom is DNA or RNA.the colour differs from almost totally translucent until white.it depens how much carefull you were when taking the upper liquid.it becomes more whiter when you have more contamination from protein carbagge. however it can still work. u should find its clearance and it may be ok for the next procedure u need it.u can also do again the last 2 steps of isolation starting from the treatment to pull down the proteins
DNA or RNA pellet can sometimes be white or translucent, depending on level of proteins contaminants as well as concentration of DNA/RNA. It is great if you get a translucent/milky pellet, which would indicate cleanliness but also less DNA/RNA, but... depending on what you are going to use the DNA or RNA, the spot at the bottom is a good sign and you should assume you have DNA/RNAproduct and go through with the next step of your experiment. It doesn't necessarily mean protein.
In my experience in doing things like PCR, cloning, RNA probes, etc, I've never had issue with white versus clear spot. If I got a spot at the bottom, it worked regardless of color, concentration, etc.. Unless you're doing something very specific that requires super clean DNA/RNA, If you have a spot, you should be fine.
Hi
I assume this will be for PCR purposes? I do not have any experience in DNA extraction. But for RNA extraction, after separation + centrifugation you should have a clear upper phase which is your RNA. If you pipette this carefully without touching you DNA viscous phase you can be sure you pellet will be entirely RNA. Depending on cell line, quality of pipetting and extraction, your RNA pellets can be white or transparent. If you use a lot of cells then is probably your pellet will be whiter. If you are working for example with tissue derived cells, then this will be trickier and you may experience a some loss.
The best way to check is by nanodrop. A good ratio 260/280 will ensure you have a good RNA quality. if the purity is below 1.8 or 1.6, then you will have for sure phenol contaminations or traces from DNA.
Good Morning Ariza, I have processed numerous types of tissues/cells, stored numerous ways (snap frozen, frozen in DMSO/cell culture medium, FFPE, OCT etc..), using multiple methods (columns, Trizol, Ribozol etc..) for over 20 years. And yes your mentor is correct, the pellet can be clear, milky in color, and on some rare occasions pure white, in usually when there is a very high concentration of RNA/DNA. I agree with Noelia on using the nanodrop for checking purity, and John on pellet color with respect to PCR (and even qPCR).
Nucleic acids should not look opaque. On the contrary, a DNA/RNA pellet should be beige in colour. And once the pellet is dry, it should be almost translucent. If there is salt contamination, when you dissolve the pellet, you can see the salt particles and get rid of them by EtOH precipitation. If you are isolating nucleic acids from plants; then the white pellet is likely to be carbohydrate, some of which are insoluble in watery solutions e.g. water, TE buffer, Tris-HCl. If this is the case, you can centrifuge the sample at high speed and 4 degrees and transfer the supernatant into a new tube. Impurities will reside as a pellet at the bottom of the tube.
Hi Ariza
Go by positive thoughts all the time in your research
For further study to check is by nanodrop. A good ratio(~2.0) 260/280 will ensure you have a good RNA quality. if the it is below ~1.8 or 1.6, then you will have for sure phenol contamination or traces from DNA.
All the best.
The white spot is the thing you want to remove. The dry DNA/RNA should be translucent. You can see something on the bottom without color when you have good extraction. It means if you extracted with quite a few amount of sample, you won't see anything. If you got a lot DNA/RNA, it looks like small amout of jelly-like stuff at the bottom with irregular shape. Not matter what, you should check with nanodrop.
I also experience the same white Pellets but I confirm it was DNA after checking through nano drop.
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