Generi Biotech - Compounds and Services for Molecular Biology

• Quantitative PCR
  • Sets for Gene Expression
  • qPCR sets for reference genes
  • Custom-Made qPCR Sets
  • Probe Master Mix
  • Standards for qPCR
  • Internal Positive Controls
• Sequencing
  • Standard Sequencing
  • 16S/ITS Sequencing
• Molecular Cloning
  • Custom cloning
  • Plasmid Amplification and Isolation
  • Competent Bacteria
• Cell Culture Applications
  • Recombinant Proteins
  • Recombinant Human SHBG
  • Cytotoxicity Testing
  • Luminescence Solution
  • Mycoplasma Detection
• Biological Identification
  • Cell Line Authentication
  • Paternity Outsourcing

Requirements for PCR Product Quality

PCR product supplied by the customer is used either to prepare the PCR standard or to perform sequencing of the product itself. In order to provide both services properly, the following information about the PCR product is necessary:

• Specification of the polymerase using which the product was amplified (must be known especially for the standard preparation)
• Photograph after electrophoresis (including the length marker and its specification)
• Whether the product was purified using some purification kit

The photograph taken after electrophoresis is used to estimate the PCR product quantity. The electrophoresis concerns an aliquot part of already isolated and purified sample on the agarose gel. 3-5 µl of PCR product is usually used, with such a quantity of the length marker that makes it possible to read DNA concentration according to instructions of the manufacturer.

Should you be interested, additional steps can be done with the PCR product. They usually represent purification or PCR product isolation from the gel. Complete amplification of the product can even be provided when the customer supplies us with primers or their sequences. The template DNA (cDNA) must also be supplied in cases when sequence of other than human, rat or mouse organism is considered.

All steps performed by us that are necessary to make sure that the PCR product shows appropriate quality for cloning or sequencing are charged at an extra price. Therefore the order needs to be consulted in advance in such cases.

Some of the requirements above are demonstrated by the following examples:

Fig. 1: Estimate of the PCR product concentration. 4 µl of the sample (upon isolation and purification) and 0.5 µg of the DNA length marker were applied. Sufficient concentration: Lanes 1, 2, 4 and 8. Threshold concentration: Lane 5. Insufficient concentration: Lanes 3, 6 and 7. This is the photograph we are interested in above all!

Fig. 2: Electrophoresis of the PCR product upon amplification under various conditions. For further purposes, it is most convenient to isolate the product in the lane 4.

Figs. 3 and 4: Electrophoresis of a seemingly homogeneous, thick strip of the PCR product. It must be ensured that the strips on the gel are divided in good quality and that the electrophoresis time is not shortened without a reason; otherwise isolation of a heterogeneous sample may threaten to occur.