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Effective date: September 2017 For Research Use Only. Not for use in diagnostic procedures.
KAPA Taq EXtra HotStart ReadyMix PCR Kit Technical Data Sheet
KAPA Taq EXtra HotStart PCR Protocol Step 2: Set up individual reactions
KAPA Taq EXtra HotStart ReadyMix can be used to replace NOTE: Always invert and/or pipette mix input material
any commercial Taq DNA polymerase in an existing for long-range PCR so as not to damage the DNA. Avoid
protocol to improve yield and/or fidelity. It can also be vortexing where possible before addition to the reaction
used to amplify mid- to long-range targets (up to 15 kb). mix.
Template DNA quality is critical to ensure successful long-
• Transfer the appropriate volume of PCR master mix,
range amplification.
template and primer to individual PCR tubes/wells of
a PCR plate.
Step 1: Prepare the PCR master mix
• Cap or seal individual reactions, mix and centrifuge
• Ensure that all reagents are properly thawed and mixed. briefly.
• Prepare a PCR master mix containing the appropriate
volume of all reaction components common to all or a
Step 3: Run the PCR
subset of reactions to be performed.
• Perform PCR with the following cycling protocol:
• Calculate the required volumes of each component
based on the following table:
Step Temperature Duration Cycles
25 µL
Component Final conc. Initial denaturation 95ºC 3 min1 1
reaction1
2
A final MgCl2 concentration of 2 mM is sufficient for most standard applications. For 3
An annealing temperature 5°C lower than the calculated melting temperature
assays that do not perform well with 2 mM MgCl2, the optimal MgCl2 concentration (Tm) of the primer pair is recommended as a first approach. If low yields and/or
for each primer/template combination should be determined empirically. non-specific amplification is obtained, an annealing temperature gradient PCR is
recommended to determine the optimal annealing temperature of the primer pair.
3
≤250 ng for genomic DNA; ≤25 ng for less complex DNA (e.g. plasmid, lambda). 4
For long-range targets, extend at 68°C.
6
Final extension should be included if PCR products are to be cloned into TA
cloning vectors.
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