General Protocol

KAPA HiFi DNA Polymerase is a novel, single-enzyme system that exhibits industry-leading fidelity and performance when compared with other proofreading polymerases and polymerase blends. KAPA HiFi has been engineered to have an increased affinity for DNA resulting in significant improvements to yield, sensitivity, speed, and the ability to amplify difficult templates. Strong proofreading activity results in an error rate 100X more accurate than Taq Polymerase.

Apllications

KAPA HiFi PCR Kits are ideally suited for routine, high-fidelity PCR including:

• Amplification of long DNA fragments for conventional sequencing (direct sequencing or sequencing of cloned PCR products).
• Amplification of short AT- and GC-rich DNA fragments for next-generation sequencing (e.g. target enrichment for resequencing).
• Amplification of DNA fragments up to 15 kb from genomic DNA or up to 18 kb from less complex targets (e.g. plasmid or lambda DNA), for cloning and protein expression or genomic characterization.
• Introduction of single or multiple point mutations using site-directed mutagenesis.
  

1.Reaction setup

Component	Final concentration	25 µl reaction
PCR Water		Up to 25 µl
Forward Primer (10 μM)	0.3μM	0.75μl
Reverse Primer (10 μM)	0.3μM	0.75μl
5X KAPA HiFi Fidelity or GC Buffer (contains 2.0 mM Mg2+ at 1X)	1X	5.0 µl
KAPA dNTP Mix (10 mM for each)	0.3 mM for each	0.75 µl
DMSO (100%) (for amplicons with a GC content >70%)	5%	1.25 µl
Template DNA	As required	10-100 ng for genomic DNA 1-10 ng for less complex DNA
KAPA HiFi DNA Polymerase (1 µ/µl)	0.5 U/ 25 µl reaction	0.50 µl

2. Cycling parameters

Step	Time and Temperature	Cycles
Initial denaturation	2-5 min at 95ºC	1
Denaturation	20 sec at 98ºC	15-35
Annealing	15 sec at optimal Th
Extension	15-60 sec/kb à 72ºC
Final extension	1-5 min/kb à 72ºC	1
Hold	4-10°C	1

3.  Products
 

Description	Size	Catalog#
KAPA HiFi DNA polymerase	100 U	KK2101
	250 U	KK2102
KAPA HiFi HotStart DNA polymerase	100 U	KK2501
	250 U	KK2502
KAPA HiFi HotStart Readymix DNA polymerase	100 rxn	KK2601
	500 rxn	KK2602

4. Results

Amplification of hgDNA targets up to 11 kb. Each target was amplified from a descending range of template hgDNA concentrations (50 ng to 0.5 ng per reaction). Reactions (25 µl each) were performed using standard 3-step cycling profiles (35 cycles): 20 sec denaturation, 15 sec annealing, and 30 sec/kb extension time. Total reaction time for the 11 kb amplicon was 3h 50mins. 12.5 µl of each reaction was loaded on the gel.