( Just before cycling pipette 1...)TripleMaster PCR System

Just before cycling, pipette 10 l of Mastermix 1 and then 40 l of Mastermix 2 into a 0.2 ml PCR tube on ice. Mix gently by pipetting 3-4 times up and down. Do not vortex and/or centrifuge!

Place immediately into a thermal cycler which is pre-heated at 93C and start cycling. Examples of cycling profiles for various long targets are given for the Eppendorf Mastercycler (Table 4). For customer-specific targets, only the annealing temperature should be adapted. By changing the time of the elongation step the product yield can be optimized following the rules given in Table 4 for different target sizes and template complexity.

Analyze large amplification products on a 0.4 % agarose gel preferably in TAE-buffer using appropriate DNA markers. For separation of products > 25 kb in a standard electrophoresis chamber, a 12-hour run at 40 V is recommended. Generally, efficient separation of very large DNA fragments is achieved only by inverse or pulse-field
electrophoresis.

Table 2: Mastermix composition for long range PCR ¹ components included in the TripleMaster PCR System Plus
² some applications may require an extra addition of Magnesium Solution (supplied) to set up a final concentration >2.5 mM, e.g. when DNA amounts >0.5 g are used.

Cycling program for long range PCR

The complete program for the 40 kb control amplification included in the TripleMaster PCR System Plus on the Eppendorf Mastercycler is given in Table 3. This data should be used as a guide for customized adaptation of the cycling program to an appropriate prim
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