I have extracted DNA from different algal samples and after extraction of DNA i used various primers for DNA amplification. I got some good results (as clear & distinct bands) but for some I see a trail of the amplified DNA. Any suggestions what might have went wrong?
1) check quality of your DNA
2) do not overload your PCR reactions - try loading less DNA. The background smear may be overloaded DNA
3) you may have either degraded or partially amplified products. Partially amplified products can arise as a consequence of overloading and the reagents become rate-limiting leading to partial amplification. An excessive number of PCR cycles will have the same effect i.e too much product in the late cycles to get complete amplification
4) you may need to adjust your primer annealing temp. to get better amplification. If one primer anneals better than the other then you may get unequal extension from each of the primers and an accumulation of different sized products which will look like a smear.
1) check quality of your DNA
2) do not overload your PCR reactions - try loading less DNA. The background smear may be overloaded DNA
3) you may have either degraded or partially amplified products. Partially amplified products can arise as a consequence of overloading and the reagents become rate-limiting leading to partial amplification. An excessive number of PCR cycles will have the same effect i.e too much product in the late cycles to get complete amplification
4) you may need to adjust your primer annealing temp. to get better amplification. If one primer anneals better than the other then you may get unequal extension from each of the primers and an accumulation of different sized products which will look like a smear.
i used reaction volume -15µl and in which i used 3µl of DNA. is it more? quality was good, (A 260/280= 1.88), amazingly enough i got good bands on the gel with other primers on same DNA content.
I was referring to the amount of DNA. The ratios look good to me. You may find that if you do several different dilutions of your DNA you will get a better signal to noise ratio.
Remember that with PCR you can get signals from a single cells worth of DNA. Most of the time we use many thousands times more DNA than that.
Dear Digvijay Singh
3µl of DNA sample is too much for 15µl reaction. Use 0.3-0.5 µl from 1.88 (A 260/280). that will be more than enough.
Digvijay Singh
Try doing a dilution of your extracted DNA, I usually do a 1:20 dilution when I extract total DNA from plant samples prior to PCR.
The fact that you got good bands using other primers then it seems like you should take a look at the annealing temp used for your PCR reaction
Usual concentration of genomic DNA for a PCR is 100 ng/ul of which most would add 1 ul to a 50 ul reaction mix so 3 ul is far too much.
For the trail you are seeing, try diluting your DNA but if you don't see this in all your samples then it is not directly linked to your DNA concentration. Check the Tms of your primers which give you the smear. If they are higher than the other sets of primers, try increasing the annealing temperature to make the conditions more stringent.
Have you tried blasting your primers to ensure they don't have homology anywhere else in the genome? This could result in a smear forming.
For genomic DNA template, I generally use 0.1 ~ 5 ng/ul (the final concentration) in a PCR reaction and it works very well for me. For plasmid DNA I use generally much less (not more than 2 ng/ul). The concentration is calculated based on Nanodrop reading of DNA extracts. From my experience, a reliable Nanodrop reading need to have good ratios of both A260/280 ( >1.8) and A260/230 ( at least >1.5), such that a good DNA extract will give a peak at 260nm using Nanodrop.
You can use Covaris Adaptive Focused Acoustics (AFA) technology. It is very innovative tools required to shear DNA and RNA without GC bias or thermal damage.
Dear Digvijay,
I would suggest you to recheck the quality of the DNA samples which you found sheared results after amplification.How old are those DNA ?Sometimes there are chances of nuclease formation which overtime degrades the DNA.Try everything from fresh like quantifying in nanodrop, gelelectrophoresis with fresh buffer and see the quality of the DNA.If everything seems to be fine then run you PCR with the primers of interest.This will hopefully help to cross check your results and hinderances.
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