Expand™ Long Template PCR System Protocol & Troubleshooting

Product No. ELONG-RO

Protocol

AMPLIFICATION OF MITOCHONDRIAL DNA

For amplification of mitochondrial DNA using the Expand Long Template PCR System refer to the following articles:

  • Paul et al., Rapid mapping of mitochondrial DNA deletions by large-fragment PCR, Trends in Genetics Vol 12, p131, 1996
  • Ray et al., The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J. Invest. Dermat., Vol 115, p 647, 2000

Troubleshooting

BUFFERS

Using the Expand Long Template PCR System, always thaw and equilibrate all buffers at +37 to+ 56 °C before use. In addition, buffers should be vortexed thoroughly. When crystals have formed, incubate at +37 to +56 °C until they are dissolved. After this step, do not place the buffers on ice. After pipetting the last reaction component, start the reactions immediately. Do not store complete reaction mixes on ice.

THERMAL BLOCK CYCLER

The thermal profile recommended in the package insert was developed for the Applied Biosystems GeneAmp PCR System 9600. Other thermal block cyclers may require a slightly different profile. Long range PCR in general is sensitive to even minute differences between ramping or heat transfer rates of different thermal block cyclers. Therefore, always develop and run your Expand Long Template PCR experiment on the same thermal block cycler. If you switch to a different thermal block cycler, adjust the reaction conditions and thermal profile. The optimal annealing temperature depends on the melting temperature of the primers and the system used. The elongation time depends on the fragment length. For a 15 kb fragment we would recommend an elongation time of 11 min.

SMEARED PCR GEL ELECTROPHORESIS PRODUCTS

When the PCR product produces a smear after gel electrophoresis, do the following:

  • Decrease number of PCR cycles
  • Decrease concentration of template
  • Reduce amount of enzyme in reaction
  • Check performance of PCR primers by amplifying a control template that has performed well in previous PCR
  • Test all three reaction buffers even if you already established the assay with one buffer: Buffer 1 - 0.5-9 kb; Buffer 2 - 9-12 kb; Buffer 3 - > 12 kb
  • In some cases, results can be improved by lowering the elongation temperature to +66 °C

SECOND GENERATION PRODUCT AVAILABLE

To meet the challenges of long range PCR we have developed the second generation kit, the Expand Long Range dNTPack. This kit includes the same enzyme mix, but provides more flexibility:

The Expand Long Range dNTPack is provided with two universal buffers, one with MgCl2 and one without MgCl2. A MgCl2 stock solution and DMSO are included as separate reagents. For best results, follow the optimization guidelines described in the corresponding package insert.

Materials

     

EXPAND is a trademark of Roche

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