- Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
- The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
- Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
- Mark the PCR tubes on lid and side and place in thermocycler.
- The annealing temperature should match the primer melting temperatures. This can be hard as Tm predictors tend to disagree.
- Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).
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