I have transformed my cloned plasmid DNA into bacteria and cultured bacteria. Then, I extracted DNA from bacteria and run in 0.5% agarose gel. But I am not seeing any DNA band except ladder.
(In nanodrop, the conc. was 86ng/microliter, but 260/280 ratio was 1.62)
To check the gel, I had run another DNA besides this DNA. But I found the band of another DNA.
What could be the problem?
Do you think 0.5% gel is to low of a percent? For my application, I use a 3% or even 4% agarose gel. Also check your voltage. Has this specific protocol worked before? I am wondering at 0.5% (assuming your fragments are not huge) maybe your bands went right out of the gel.
Maybe something went wrong with your PCR.
@Andrey, sorry, I didn't save the picture. Because, I can't see any band of that DNA except ladder
@Andrew, this Protocol worked before perfectly. For confirmation, I loaded another DNA in another well. But it can be seen.
It seems the concentration of the gel is low. Even the DNA concentration is also pretty low. I would suggest you to increase the concentrations of both the gel and the DNA and also make sure you use a stable low voltage for 30-40 minutes. Optimize the conditions. After all these hopefully you will be getting a good results.
How much DNA did you load.
What was the size of the band(s) did you expect. Was the DNA circular, supercoiled, or linear.
Was everything supposed to be in one band or were you looking at fragments.
Also if you ran a control ladder, could you see the band that corresponded to the size of your DNA? Were you looking at supercoiled DNA, but using a ladder that was linear DNA, because they do not run in the same positions.
Suggestions:
Unless you are looking at very large pieces of linear DNA, I would suggest using a higher % between 1 and 2%.
Run a control lane with a known amount of your un-modified plasmid. If you cannot see that there is another problem.
Also your 260/280 ratio was poor. Should be between 18.-2.0. If you have a lot of contamination with protein, it may be conjugating to your DNA which would cause a smear if you have enough DNA to actually see. I usually try for a couple of 100 ng of DNA in a given band.
@Josias: Hi, I loaded 6microlitre of DNA (1mcl DNA, 1mcl H2O, 4mcl 6X LD buffer). No, DNA was in the well. Because, I can see other DNA's band.
@Ranganathan: Hello, thank you very much for your suggestion.
@George: I loaded 6microlitre of DNA (1mcl DNA, 1mcl H2O, 4mcl 6X LD buffer). No, DNA was in the well. Because, I can see other DNA's band. I am looking for circular DNA. After recombinant plasmid cloning and transformation, I grew bacteria. But after extraction of the plasmid DNA from bacteria, I can't see in the gel.
Hi, I think you should use more amount of DNA, maybe 5 micro-liter, don't add water to it, just add loading buffer
Check this page
https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-electrophoresis-troubleshooting.html
Rahul Mallick and everyone getting a low yield of DNA - it could be due to contamination
260/280 ratio of ~1.8 is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
Below a few tips from the recent experience of one of my students:
1. Check transfection medium for mold or other contamination (mold grows in medium with antibiotic)
2. Make sure LB-Agar is not too hot when adding antibiotic to make plates (antibiotic could be destroyed, and bacteria without environmental pressure will lose plasmid, and they will not grow in liquid culture)
3. Shake bacteria for long enough in LB-antibiotic liquid culture (we use 220 rpm for 14-18h). It is good to have one extra tube of LB-antibiotic without any inoculation to check for possible contamination.
Standard miniprep using above growth conditions gives us around 20 ug DNA (100 ul of DNA with a concentration of 200 ng/ul )
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