How can we prevent contamination in gel electrophoresis?

How can we prevent contamination in gel electrophoresis?

To avoid future contamination, you should do everything above and a few more things:

1. Work in dedicated space.
2. Store PCR reagents and PCR products separately.
3. Aliquot.
4. Store PCR tubes/tips/racks separately.
5. Don’t flick your tubes open.
6. Use a master mixer and add your template last.
7. Train others.

How does contamination affect gel electrophoresis?

Contamination of the Sample One source of error is contamination of the DNA sample. If there is foreign DNA in the sample, the gel will have more bands than would be found in a gel that contains only the purified sample.

What are the possible sources of contamination when performing PCR?

The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards.

How does contamination affect PCR?

PCR product carryover contamination. The most important source of contamination is from the repeated amplification of the same target sequence, which leads to accumulation of amplification products in the laboratory environment. Even minute amounts of carryover can lead to false-positive results.

How does PCR prevent cross contamination?

Separating pre- and post-amplification areas is key to preventing contamination. Prepare your PCR master mix in a template-free room (see next bullet), using reagents that never come into contact with potential sources of contamination. Maintain a separate area for analyzing PCR amplicons.

What are common errors when doing gel electrophoresis?

Proteases that act at room temperature upon proteins in the sample buffer prior to heating, cleavage of the Asp-Pro bond upon prolonged heating of proteins at high temperatures, contamination of sample or sample buffer with keratin, leaching of chemicals from disposable plastic ware, and contamination of urea with …

What causes fuzzy bands in gel electrophoresis?

The bright fuzzy bands at the bottom of the gel are typical of those caused by primer dimers. Primer-dimers can be minimized by using hot start PCR or by using a higher annealing temperature.

How do you clean PCR products?

Classical methods used to clean up PCR products prior to sequencing include gel electrophoresis, ethanol precipitation, and column chromatography. While quite effective, these methods can quickly become cumbersome when processing multiple samples in parallel and exhibit varying degrees of sample loss.

How do you contaminate DNA?

DNA evidence can be contaminated when DNA from another source gets mixed with DNA relevant to the case. This can happen when someone sneezes or coughs over the evidence or touches his/her mouth, nose, or other part of the face and then touches the area that may contain the DNA to be tested.

How do we avoid sample contamination in DNA extraction?

Use disposable instruments or clean them thoroughly before and after handling each sample. Avoid touching the area where DNA may exist. Avoid talking, sneezing, and coughing over evidence. Avoid touching your face, nose, and mouth when collecting and packaging evidence.

Can a PCR reaction show a smear in the gel?

I loaded a PCR reaction on an agarose gel and I get a long smear with my band a little more intense in the smear. If I dilute the DNA I obtain my band but not perfectly clean from a sample.

How can you tell if you have PCR contamination?

So, don’t become over confident and don’t justify your laziness, just run your negative controls because THAT is how you will know if you have contamination. A PCR negative control is usually just the normal PCR master mix (polymerase, primers, buffer, nucleotides) but instead of adding template, you add water.

Where to store PCR reagents and PCR products?

Setup your PCR away from where you analyze PCR results. This is best done in a hood or, at minimum, benches away from where you run gels. Store PCR reagents and PCR products separately. In the same way that you do not want your PCR analysis to be near where you setup, you should never store PCR product and reagents together.

Which is the biggest source of PCR contamination?

The biggest source of PCR contamination is aerosolized PCR products, which are created when you open a tube or pipette amplified PCR product. Once these droplets are created, being small, they travel well. These tiny droplets easily spread all over your bench and equipment, where they can find their way into your next PCR and be amplified.