I'm in an introductory cell and molecular biology lab. This is a DNA gel electrophoresis. I've read that degraded DNA is indicated by smears (column 3) and also by a tight high molecular weight band (column 4). I'm confused which is the right answer.
Thanks in advance.
Line 3 seems right: DNA degradation (usually due to an endonuclease present in the prep) results with a population of fragments of various lengths which look like a smear.
Line 3 seems right: DNA degradation (usually due to an endonuclease present in the prep) results with a population of fragments of various lengths which look like a smear.
As Ana mentions line 3 looks like degraded DNA, while line 4 looks like DNA with a band of contaminating RNA
It looks as though lane 3 indicates degraded DNA. This is a helpful video that explains gel electrophoresis and how to analyze data for future questions! https://www.youtube.com/watch?v=EK3ENBm5azg
Yes, stream 3 not showing clear bands as others that's why the regarded DNA lie in that region.
Lane 3 tells you about the quality of your DNA. Please you can also check the concentration through nanodrop
Of course, lane 3 is the smear of degraded DNA as a result of random digestion of genomic DNA that you can see just in lane 3.
Mira Mzayek Following DNA extraction, I suggest you to carry out two steps: nanodrop quantification, electrophoresis gel visualization and if possible qubit quantification. Regarding the interpretation of the agarose gel: lane 1 would be a good DNA band, at the lane 2 would be correct, although it has less DNA concentration. The 3 is DNA degraded since there are no clear bands and usually appears throughout the column. For the lane 4 I consider that would be contamination with RNA (this is confirmed with the nanodrop values). Another option is that you see several bands that would be that the DNA is fragmented, or that there are plasmids. Also, there is at the market to quantify ADN special marker.
If you would have shown the nanodrop/Qubit readings it would have been much better. Anyway from the gel image, lane 3 is clearly degraded. While in the lane 4, the second band at the bottom represents RNA contamination. @ Mira Mzayek
yes I think the third due to the pool of fragments of different lengths. The fourth one may recall a contamination beside DNA or simple a different kind of DNA (different shapes, length..). I think the thickness of the stripe in fourth one , does not mean a smear like the third one, but simply a high quantity of Dna of the same length, but I do not how it could affect following steps of purification .
Please correct me for wrong supposition .
I agree, lane 3 is randomly degraded DNA. To check the quality of your DNA, I would suggest, like some before me, Qubit quantification and gel electrophoresis... the nanodrop is not reliable enough if your downstream analysis do not include PCR... it does detect all the nucleic acids in the sample..whether degraded, high quality, DNA, RNA etc....
Well 3 has degraded DNA. DNA might be degraded while you are extracting DNA. Well 4 has contaminations.
Mira Mzayek
Lane 3: Depeticts smeared DNA sample with a very small size of fragments, as it is traveled to the lower side of the gel, which appears as a smear on the gel. DNA degradation usually due to an endonuclease present in the prep.
Lane 4: Intact band near the well is your sample DNA and another band present in the lower side of the gel which is RNA contamination.
Lane 3, the degraded one. Also, lane 4 has contamination. The degradation may be due to many reasons: 1. Traces of endonuclease remain in the DNA during purification. 2. Keeping the DNA sample in R.T for a long time. 3. Repeated freezing and thawing. 4. Using DNA extracted from formalin-fixed paraffin-embedded samples. 5. Exposing DNA samples to heat or physical shearing.
Column 3 is a pool of degraded DNA while column 4 a small thick band of DNA which is contaminated by RNA fragments.
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