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