I have isolated DNA from bacterial culture.I amplified 16s DNA with PCR. But I am getting smearing after PCR. What could be the problem regarding this? I am attaching photo of my gel.
Hi, here are some possible reasons:
Non-optimal Mg++ concentration
Nucleotide concentration is too high or unbalanced
DNA contamination / carry-over
Primer annealing temperature is too low
Mispriming caused by secondary structure of template, snapback, or excessive homology at 3' ends of primers
DNase activity (indicated by smears visible on gel below expected band size)
Incorrect template to enzyme ratio
Cheers, Nadine
In my experience, the smears on gels after PCR most often comes from the DNA (template) overload. Try diminishing the amount of loaded template DNA in increaments of 0.5 in a series of reactions. If that doesn't help, all parameters mentioned by Nadine are to be optimized.
hay Brijesh, how does ur DNA look on gel i mean does it look like smear r sharp band,,,,chek it out n if it is ok on gel then use minimum amount of DNA for pcr...may be you u r using too much of template ..n make sure u have treated ur dna with RNAse A....
use small amount of template, use low concentration of template,.reduce the tendency of contamination after isolation of dna,.use Dnase-free pcr tube,.reduce the inhibitor that may be present in isolated bacterial dna, e.g EDTA. reduce the cycle of pcr reaction if getting smear for the first time by using the same amount of template.
Smearing appears due to contamination in DNA in some cases. Minimize the annealing temperature to overcome the problem.
1. RNA contamination (forgot the treatment with RNAseA).
2. DNA overload (measure DNA concentration and take no more than 50 ng).
3. Unspecific amplification (optimize your PCR as was recommended in previous answers).
As it's mentioned above, I would suggest you using less amount of template DNA. If you got no band after the PCR you can switch to nested PCR. Run your first round of PCR with low amount of DNA and outer primers then do PCR purification on your sample and take your template for secound round with inner primers!
Maybe you load high concentrated PCR product. Dilute your PCR product with loading dye.
Smearing the PCR product due to the high concentration DNA template should not be the case. High concentration of DNA template would cause very thick bands on the gel, unless your DNA degraded or RNA contaminated.
As suggested by many use min amount of template DNA. Make sure that your primer is pure enough without any other oligonucleotides
Dear Brijesh. I am also facing the same problem.Can u please let me know how you rectified it?
DNA contamination, RNA in DNA sample, hight concentration of DNA in the PCR reaction can cause smearing.
Recently I've tried ethanol precipitation of PCR product, and I've got pretty clean pictures on the gel.
Dear Sara Ohadi let me know the ethanol precipitation protocol.
Here is the method that worked for me:
Add 5µl of 3M Sodium Acetate (pH 4.6) and 50µl of 95% ethanol to 50µl of your PCR product. Mix them well by gently inversion for few minutes. Spin for 20 minutes at maximum speed. Aspirate off the supernatant and rinse the pellet with 70% ethanol (again you can spin down the pellet and remove the 70% ethanol). Elute the PCR product pellet in water.
DNA smearing usually caused in plants due to high concentration of template DNA. So please see after reduction of the template. Another important thing while you are running the gel the buffer should be totally above the agarose gel.
Theres many reasons to smearing on the gel, but for looking at PCR alone. Are your primers high in GC content? If so you could use a specific buffer (5x GC buffer from NEB comes with dna phusion polymerase). Is your annealing temperature the right one? Did you extract your plasmid from BL21? BL21 usually has a lot of endonucleases even after gene extraction.
Hi, Dr. Brijesh Dabhi
Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:
- too much starting template Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
- carry-over contamination If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
- enzyme concentration too high When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.
- too many PCR cycles Reduce the number of cycles in steps of 3 cycles.
- Mg2+ concentration not optimal
- Badges
- Science method