What causes extra bands in gel electrophoresis?

What causes extra bands in gel electrophoresis?

Typically, off-target DNA bands are caused by either partial digestion or Star Activity. You need to compare your digestion to the expected DNA banding pattern. In this case, you may need to purify the DNA to remove any contaminants, use more enzyme and/or increase the incubation time to ensure complete digestion.

What does a thick band in gel electrophoresis mean?

Thicker bands in gel electrophoresis mean there is more of that particular size molecule in the sample.

Why are there 3 bands in gel electrophoresis?

Band 3 contains smaller DNA fragments than band 2, but is still much brighter. This is because there is more (nanograms of) DNA in 3 than in 2 (the number of molecules in 3 must be much higher than in 2).

What does incomplete digestion look like on a gel?

Incomplete digestion results in additional bands above the expected bands on a gel. These bands disappear when the incubation time or amount of enzyme is increased, as seen when comparing sample in lanes 2 and 3 to the completely digested sample in lane 4 (Figure 8).

Why do multiple bands appear in a DNA gel?

Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.

Why are some DNA bands thicker?

You may have noticed that some of your bands are thicker and darker, whereas others are thinner and lighter. A thicker, darker band does, as you might expect, mean that there is more DNA present, but this is not because you have more of that DNA in you!

What causes the banding pattern to form on the gel?

DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size. They will appear as bands on the gel.

What are bands in gel electrophoresis?

A well-defined “line” of DNA on a gel is called a band. Each band contains a large number of DNA fragments of the same size that have all traveled as a group to the same position. A single DNA fragment (or even a small group of DNA fragments) would not be visible by itself on a gel.

Can I digest PCR product directly?

Whatever is your PCR product, you could in principle digest it, but the PCR buffer will not allow you to do so, you’ll need to substitute with a specific RE buffer.

What would you expect to see if the digestion was incomplete?

You need to compare your digestion to the expected DNA banding pattern. 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).

How to interpret DNA gel electrophoresis results?

Gel Electrophoresis. Lane 1: DNA Ladder. Lane 2: Undigested plasmid A. Lane 3: Completely digested plasmid A. For Lane 2, you may be able to see two bands. The faint band on top is OC and the one below is the CCC form. For the Lane 3, it’s the completely digested plasmid, so the band has a linear form.

Is the EcoRV gene the same as the Hf gene?

EcoRV-HF® has the same specificity as EcoRV (NEB #R0195), but it has been engineered for reduced star activity. Cleaves to leave a ligatable blunt end in the tetracycline resistance gene of pBR322. Impaired by some combinations of overlapping CpG methylation.

Which is the high fidelity version of EcoRV?

EcoRV has a High Fidelity version EcoRV-HF ® (NEB #R3195). High Fidelity (HF) Restriction Enzymes have 100% activity in rCutSmart Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity.

What does DNA gel look like under a microscope?

Smaller DNA fragments can move quickly through the pores, while larger fragments get caught and therefore travel slowly. Let’s look at how this all works. Under a powerful microscope, a gel will look porous, but to the naked eye, it looks like a smooth, opaque gelatin in the shape of a square with wells near one end of the surface.