Troubleshooting guides

Troubleshooting guides

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How long does it take to complete the DNA/RNA extraction protocol?

It will depend on the number of samples you intend to use. Usually for 5-10 samples, 60 min is enough to perform the entire protocol. Before starting, be sure that all reagents and solutions are prepared.

What lysis method should I use to obtain DNA/RNA from my sample?

The lysis method that should be used will depend on the membrane and cell wall properties of the cells. If the cell does not have cell wall, you can use the lysis buffer only. Although if the cells have cell wall, we strongly recommend the use of enzymatic or mechanical processes to disrupt the membrane and cell wall, such as lyticase or beads, respectively.

What is the range of concentration expected in a DNA/RNA extraction protocol?

The DNA concentration will depend on the amount of DNA/RNA contained in cell sample. Usually 1-5µg is obtained from a typical DNA/RNA extraction protocol.

Is it possible to extract DNA/RNA from a small volume of cell sample?

Yes, it is. However, you should expect DNA concentrations to be less than 1 μg of DNA.

Can I extract DNA/RNA from solid samples?

When you are using biological samples to perform DNA/RNA extraction you should add a previous step in the extraction protocol. Usually, it is recommended to use liquid nitrogen to break the solid sample into small volumes in order to obtain a cell suspension after resuspension buffer is added.

What are the quality standards for DNA/RNA?

When you check the DNA/RNA purity in Nanodrop you should expect to obtain A260/280 ratios around 2.1 and 1.8, for RNA and DNA respectively. The A260/230 ratio should be close to 2 and never below 1.8.

How long can I store the DNA/RNA?

Depending on your storage conditions, you can store at 4°C for immediate use or −20°C for 1-2 months.

Is it possible to perform a PCR reaction for different genes with primers that have different Tm?

It is possible. You just have to use the temperature gradient setting of the thermocycler and place the PCR tubes in the correct temperature row or column, depending on the thermocycler features.

Should I use the same polymerase for any PCR?

A huge number of commercial polymerases are available, and each one has different properties and applications. You should take into account what will be the main goal of your PCR reaction.

Can I use PCR products for molecular cloning?

One of the applications of PCR is to generate significant amounts of a specific DNA fragment to perform molecular cloning. However, it is recommended to perform a PCR cleanup before using the PCR product. PCR reagents can inhibit some enzymes used in molecular cloning.

Is it required to always perform DNA extraction before PCR?

In some situations, you do not need to perform DNA extraction, which is the case of colony PCR. Usually, colony PCR is used to check if a specific fragment is present in a sample without DNA extraction being done, typically after molecular cloning. This approach uses intact cells previously exposed to microwaves (which makes the cell membrane more permeable), and then the PCR reagents are added.

Can I use PCR to generate fragments flanked by restriction sites?

If you will use the PCR product for molecular cloning, you can choose which restriction enzymes you want to use and then add to the forward and reverse primer the sequence of each restriction site.

How can PCR be used in site directed mutagenesis?

The site directed mutagenesis method follows all the principles of a standard PCR, and the differences are: the set of primers should contain the nucleotide mutation to be generated; the polymerase should be a high-fidelity polymerase in order to avoid additional mutations.

Which “housekeeping genes” should be selected?

It is crucial to choose housekeeping genes that maintain the same expression levels in all the conditions tested. It is recommended to choose more than one housekeeping gene and make a preliminary test to verify that the expression levels are the same under the experimental conditions tested. Usually, these are the most commonly used housekeeping genes: 18S, GAPDH, ACTB. However, it has been shown that in some samples and conditions even these genes have different expression patterns.

Which is the right number of replicates to be used in qRT-PCR?

The experimental system chosen will define the number of replicates to be used. At minimum, it is always recommended to perform 3 replicates for each sample.

What is the sensitivity range of qRT-PCR?

The sensitivity of qRT-PCR is highly dependent on the thermocycler used and experimental conditions. Usually it is possible to detect a minimal quantity of 10-20 copies of template.

What are relative and absolute quantification?

Absolute quantification calculates the total number of a specific target fragment when compared with a standard sample with known number of copies. The relative quantification calculates expression differences between samples; usually the sample expression profile is compared with the housekeeping gene expression levels.


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