Flow Cytometry Technical Resources

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Once you have your genetic samples, one of the first steps of PCR is to design the primers required to perform the PCR reaction.

Primer Design

The first step to proceed with a standard PCR is the primer design. You should determine which fragment of the DNA template you are looking to amplify. Thus you need to know the DNA sequence of the template. The NCBI database is a web server with all DNA sequences known; you can look for your sequence there. After that, you should design two primers, the forward and the reverse primer.  Primer design is a critical step in a PCR protocol. The set of primers should flank the fragment you intend to amplify from the DNA template. The forward primer will anneal with 3’-5’ DNA strand and the reverse primer will anneal with the 5´-3’ DNA strand. Refer to the diagram below.

Reverse Forward Primer Example

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Many of the problems with PCR protocol are associated with wrong primer design. The most common mistakes in design include:

  • Primer dimers
  • Hairpin loop structures by primer self-annealing
  • Non-specific annealing

The primer length should be around 18-30 bp, the GC content close to 50% and the melting temperature (TM) between 55ºC and 65ºC. In order to calculate melting temperature, you can use the following equation Tm = 2°C(A+T) + 4°C(G+C). Although most commercial DNA polymerases provide their own calculator and specific instructions for different conditions, several web platforms for assisting with primer design are available, such as http://www.ncbi.nlm.nih.gov/tools/primer-blast/.

Once you have successfully acquired your DNA samples and designed your forward and reverse primers, the PCR experiment can proceed.

General PCR protocol

Reagents –

  • Forward and reverse primers
  • dNTPs
  • DNA template
  • DNA polymerase

Note: From the beginning of your PCR experiment until the end, you should always wear gloves in order to avoid DNA contamination. All the reagents, primers and enzymes should be kept in ice. Make sure that primers, DNA template, buffer are completely unfrozen before starting to prepare the PCR solution. It is important to create an experimental design in accordance with scientific guidelines, including positive control and a negative control. As the negative control, you can prepare a PCR deprived of DNA template. For the positive control you should use a set of primers and DNA template shown to work properly in previous experiments.

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Depending on the DNA polymerase you use, final concentration of each reagent will vary. These are the standard volumes and concentrations taking into account a 50 ul reaction:

  1. In PCR tubes of 200 μl:
    1. Add 38 μl sterile water
    2. Add 2 μl of forward primer (10 μM)
    3. Add 2 μl of reverse primer (10 μM)
    4. Add 1 μl of dNTPs (50 μM)
    5. Add 5 μl of reaction buffer containing MgCl2 (10X)
    6. Add 1 μl of DNA template (100 ng/μl)
    7. Add 1 μl of DNA polymerase (0.5 U/μl)
  • Note:  If you are willing to do a significant number of PCR reactions it is recommended to prepare a reaction mix, excluding the reagents that will be different from experiment to experiment (usually the DNA template or the set of primers).
  1. Pipette gently the reaction mixture to allow good homogenization.
  2. Short spin in a centrifuge is recommended.
  3. Make sure the caps of PCR tubes are closed.
  4. Insert the tube in the thermocycler.

Tip: Set up the PCR settings, such as temperature, time and cycles before your PCR reaction is ready. See the diagram below for reference of typical PCR reaction settings.

PCR Cycle Settings Graph

Note: Always pay attention to the guidelines of the DNA polymerase, since denaturation, annealing and extension temperature and time highly depend on it. In the thermocycler you should set up these steps subsequently (refer to diagram above):

  1. An initial step of DNA denaturation at 94°C to 98°C for 3 to 5 min depending on the optimal temperature of the DNA polymerase
  2. Denaturation temperature (the same used in the step before) for 30 seconds
  3. Annealing temperature taking into account the Tm of the primers for 30 seconds
  4. Extension temperature at 72°C and taking into account the size of the fragment to be amplified

These steps should be repeated for 25 to 35 rounds (cycles).  A final step of extension is required to allow all the PCR products to be correctly synthesized, usually at 72°C for 10 min. Finally, the temperature should be reduced to 4°C to store the PCR product.

PCR Product Analysis: Gel Electrophoresis

*Refer to Key Principles Section (Data Acquisition and Analysis: Gel Electrophoresis) for detailed explanation of how to analyze PCR results*

Reagents:

  • Add 5 μl of the PCR product in a new PCR tube
  • Add 1 μl of DNA loading buffer

Common DNA loading buffer (6X) recipe:

  • 30% (v/v) glycerol
  • 25% (w/v) bromophenol blue
  • 25% (w/v) xylene cyanol FF
  1. Load the 6 μl mixture in an agarose gel 1%.
  2. Load 5 μl of DNA marker in the same gel.
  3. Use an UV transilluminator to visualize the PCR product in the agarose gel.

Note: In order to avoid staining with ethidium bromide, you can use Midori or Red Safe pre stained gels, which are less toxic compounds. Use a DNA marker to compare the correct size of the PCR product.

Reverse transcription protocol

Reverse transcription is the process by which RNA is transcribed into DNA, which will allow us to perform experiments such as qRT-PCR under the catalytic activity of a specific DNA polymerase that is only able to amplify fragments from DNA molecules.

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Note: From the beginning of your PCR experiment until the end, you should always wear gloves in order to avoid DNA contamination. All the reagents, primers and enzymes should be kept in ice.

In a 200 μl PCR tube:

  • Add 2 μg of total RNA
  • Add 1 ul of oligo dt (50μM)
  • Add 1 μl of dNTP (10mM)
  • Add DEPC-treated water up to 10 μl
  1. Incubate at 65 °C for 5 min and then keep the tubes on ice for 1 min
  2. Add 2 ul of RT buffer (10X)  
  3. Add 4 μl of MgCl2
  4. Add 2 μl of DTT (0.1 M)
  5. Add 1 ul RNase inhibitor (40 U/μl)
  6. Add 1 ul of reverse transcriptase (200 U/μl)
  7. Incubate at 50°C for 60 min, followed by 85°C for 5min
  8. Afterward, keep the tube on ice
  9. Add 1 μl RNase H
  10. Incubate for 30 min at 37°C
  11. Store the PCR tube at -20°C
  12. For RNA quantification and quality use a NanoDrop equipment to measure the RNA (260 nm), protein and salt concentrations

qRT-PCR

Primer design:

Similarly to standard PCR, a set of primers should be designed as described before in order to amplify each cDNA fragment of interest, usually corresponding to a specific gene. Moreover, it is also recommended to design primers specific for other cDNA genes for data normalization procedures, these are also known as “housekeeping” genes (e.g. 18S, GAPDH, ACTB).

Note: Set up the PCR settings, such as temperature, time and cycles before your PCR reaction is ready.

For each cDNA gene, prepare the following reaction mix in 200 μl PCR tubes for a final volume of 50 μl:

  • Add 25 μl of SYBR Green Mix (2x)
  • Add 1 μl cDNA from the previous cDNA sample
  • Add 1 μl Forward Primer (10 mM)
  • Add 1 μl Reverse Primer (10 mM)
  • Add 22.5 μl DEPC Water
  • Insert the tubes in the thermocycler

When the PCR is finished, perform quantification analysis by comparative Ct method. The Ct method compares the Ct values of the samples with standards samples. The Ct values of the controls and the samples are normalized using the Ct values obtained for housekeeping genes. To validate the Ct calculation, it is required that the amplification efficiencies of the target should be close to endogenous. This can be accessed through the utilization of different template dilutions.