Expand™ Long Template PCR System Protocol Troubleshooting

The Expand™ Long Template PCR System (Product No. ELONG-RO) is an enzyme mix that contains thermostable Taq DNA polymerase and a thermostable DNA polymerase with proofreading activity. This polymerase mixture produces a high yield of PCR product from genomic DNA. The Expand™ Long Template PCR System enables amplification of DNA fragments up to 20 kb from human genomic DNA and 40 kb from λDNA.

Troubleshooting the DNA Amplification 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

Buffers
Always thaw and equilibrate all Expand™ Long Template PCR System buffers at 37-56 °C before use. In addition, buffers should be vortexed thoroughly. When crystals have formed, incubate at 37-56 °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 minor differences between ramping or heat transfer rates of different thermal block cyclers. Therefore, always develop and perform the 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 have 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. An MgCl₂ stock solution and DMSO are included as separate reagents. For best results, follow the optimization guidelines described in the corresponding package insert.