Why do we smear bands in PCR?
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. DNA contamination, RNA in DNA sample, hight concentration of DNA in the PCR reaction can cause smearing.
What causes separation of DNA bands during electrophoresis?
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.
Why are two bands usually seen after PCR and agarose gel electrophoresis?
You could try varying your primer concentrations and also do a gradient PCR with different primer concentrations. It seems like there is excess primer in your reaction mix which is why you are seeing a bright bands which maybe excess primers and lighter bands that are non-specific bands.
Why do we purify the samples after PCR?
Purification of DNA from a PCR reaction is typically necessary for downstream use, and facilitates the removal of enzymes, nucleotides, primers and buffer components. The amplified DNA remains in the aqueous layer of the mixture, along with other reaction components (e.g., buffers, nucleotides and primers).
Why do bands smear gel?
Scientists hope for clear bands, but sometimes the bands smear. This smearing is usually the result of poorly prepared gels, loading undiluted samples into the wells or poor quality samples.
Why does uncut DNA show multiple banding?
When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see 3 bands. This is due to the fact that the circular DNA takes on several conformations the most abundant being: supercoiled, relaxed and nicked. If your digest lanes look like your uncut lane then there is something wrong!
Why are there two bands in the digested sample?
Typically, off-target DNA bands are caused by either partial digestion or Star Activity. If the bands in both lanes are similar to the expected pattern and the additional bands are limited to spaces within the upper and lower bands of the expected pattern, the digestion is incomplete (partial).
Why are there 2 bands in each lane of DNA fragments?
It is clear that in lane 2 two fragments are present in non-equimolar amounts (the upper band must contain longer DNA molecules, but is less intense than the lower band..). In this particular case it’s because they represent two circular forms of the same plasmid DNA (oc on top, and ccc below).
Why run a gel after PCR?
Sometimes, more than one DNA sequence might be copied. Gel electrophoresis can be used to check whether or not this happened. This will help you to identify the band containing the DNA of interest. The DNA can then be extracted from the gel and used.
Why are there no bands in my PCR results?
Again, it is unlikely your primer concentration is to blame for complete absence of bands. Even as little as .1µM primer will be sufficient for most PCR. Template DNA: While theoretically only one molecule is needed for amplification, realistically for a typical 25 or 30 cycle PCR this may not be sufficient.
What happens if you forget a component in PCR?
Forgetting just one component of the PCR reaction, whether that be the DNA polymerase, primers or even the template DNA, will result in a failed reaction. The simplest solution is to repeat the reaction. Take your time to ensure everything has been added.
What is the positive and negative control for DNA extraction?
I use the original construct as the positive control, water, DNA extracted from untransfected cells as the negative controls. However, I keep getting bands in both negative controls. I have changed the dNTP, buffer, water, primer…etc. everything I can think of that might be contaminated. I also extracted DNA again from freshly prepared cells.
What happens if you put ethanol in a PCR?
Ethanol can be carried over from the DNA extraction process, for example. Putting these into a PCR reaction could hinder the process. If you are unable to re-extract the template, try diluting the template DNA. This will reduce the amount of PCR inhibitor in the reaction.