KAPA2G Fast Multiplex PCR Kit

This protocol its for my PhD because its difficult to find online

Step 1: Prepare the PCR master mix

• Ensure that all reagents are properly thawed and mixed. 

• Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.

• Calculate the required volumes of each component based on the following table:

Component	25 μL reaction1	Final conc.
PCR-grade water	Up to 25 μL	N/A
2X KAPA2G Fast Multiplex Mix^2^	12.5 μL	1X
10 μM Forward Primers	0.5 μL each	0.2 μM3
Template DNA4	As required	As required

1 For volumes smaller than 25 μL, scale reagents down proportionally. Reaction volumes >25 μL are not recommended, as this may result in reduced reaction efficiency.

2 KAPA2G Fast Multiplex Mix contains 3 mM MgCl2 at 1X. Reactions may be supplemented with additional MgCl2 if required.

3 When first attempting multiplex PCR with this kit, use each primer at a concentration of 0.2 μM. Depending on the results obtained, primer concentrations can then be adjusted to allow for similar yields of all fragments in the multiplex.

4 Use <100 ng genomic DNA (10–100 ng) and <1 ng less complex DNA (0.1–1 ng) per 25 μL reaction as first approach.

NOTE: For GC-rich multiplex PCR assays, reactions may be supplemented with 5–10% DMSO, or 0.5–1 M betaine. Refer to Important Parameters: Amplicon size and GC content for more information.

Step 2: Set up individual reactions

• Transfer the appropriate volumes of PCR master mix, template and primer to individual PCR tubes or wells of a PCR plate.

• Cap or seal individual reactions, mix and centrifuge briefly

Step 3: Run the PCR

• Perform PCR with the following cycling protocol:

Step	Temperature	Duration	Cycles
Initial denaturation1	95ºC	3 min	1
Denaturation	95ºC	15 sec
Annealing2	60ºC	30 sec	20–354
Extension3	72ºC	15–90 sec/kb
Final extension	72ºC	1 min/kb	1

1 Initial denaturation for 3 min at 95°C is sufficient for most applications. Use 5 min at 95°C for GC-rich targets (>70% GC content).

2 Use of the optimal annealing temperature is critical for successful multiplex PCR. Start with 60°C, and adjust up or down as required, or perform annealing temperature gradient PCR.

3 The extension time is dependent on the level of multiplexing, as well as the amplicon size. Refer to the table below for guidelines on the recommended extension time.

15–30 sec	30–60 sec	60–90 sec
Low plex	Medium plex	High plex
Up to 5 amplicons <1 kb in size	Up to 10 amplicons <1 kb in size	Up to 10 amplicons <1.5 kb in size
Up to 10 amplicons <500 bp in size	Up to 20 amplicons <500 bp in size	Up to 30 amplicons <500 bp in size
Final extension	72ºC	1 min/kb

4 The number of cycles used should be kept to a minimum to ensure an even yield of all amplicons in the multiplex. Start with 30 cycles, and adjust as required.

Post Tags  PhD

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