General protocol
KAPA™ LongRange DNA Polymerase Protocol
KAPA™ LongRange Hot Start DNA Polymerase Protocol
KAPA™ LongRange Hot Start ReadyMix with dye Protocol
KAPA LongRange Products
Kit components
- 5 U/μl KAPA Long Range Polymerase
- 5X KAPA Long Range Buffer
- 25 mM MgCl2 solution
- 250 μl KAPA dNTP Mix (10 mM each)
Quick Notes
- Denature at 94°C, increase denaturation times for fast-block instruments.
- Extend at 68°C, especially for long range/high sensitivity PCR.
- Replace Taq for Standard PCR, with no protocol change.
- Half concentration of enzyme (1.25 U/50 μl) can be used for midrange targets.
- Buffer is supplied at 5x concentration with separate magnesium for optimization.
- Products can be cloned in T-overhang cloning vectors.
Standard PCR Protocol for short targets (up to 8 kb) and/or high concentrations of template DNA
| Final Concentration | 50 μl rxn | |
| PCR grade waterup to 50 μl | – | As required |
| 5x KAPA LR buffer (without Mg2+) | 1X | 10 μl |
| MgCl2 (25 mM) | ≥1.75 mM | 3.5 μl |
| dNTPs (10 mM each dNTP) | 0.3 mM | 1.5 μl |
| Forward primer (10 μM) | 0.5 μM | 2.5 μl |
| Reverse primer (10 μM) | 0.5 μM | 2.5 μl |
| Template | As required | As required |
| KAPA LongRange DNA Polymerase (5 U/μl) | 1.25 U / 50 μl | 0.25 μl |
| Total | 50 μl |
| Final Concentration | 50 μl rxn | |
| PCR grade waterup to 50 μl | – | As required |
| 5x KAPA LR buffer (without Mg2+) | 1X | 10 μl |
| MgCl2 (25 mM) | 1.75 mM | 3.5 μl |
| dNTPs (10 mM each dNTP) | 0.3 mM | 1.5 μl |
| Forward primer (10 μM) | 0.5 μM | 2.5 μl |
| Reverse primer (10 μM) | 0.5 μM | 2.5 μl |
| Template | As required | As required |
| KAPA LongRange DNA Polymerase (5 U/μl) | 1.25 U / 50 μl | 0.25 μl |
| Total | 50 μl |
| Final Concentration | 50 μl rxn | |
| PCR grade waterup to 50 μl | – | As required |
| 5x KAPA LR buffer (without Mg2+) | 1X | 10 μl |
| MgCl2 (25 mM) | 1.75 mM | 3.5 μl |
| dNTPs (10 mM each dNTP) | 0.3 mM | 1.5 μl |
| Forward primer (10 μM) | 0.5 μM | 2.5 μl |
| Reverse primer (10 μM) | 0.5 μM | 2.5 μl |
| Template | As required | As required |
| KAPA LongRange DNA Polymerase (5 U/μl) | 1.25 U / 50 μl | 0.25 μl |
| Total | 50 μl |
Products
| Description |
Size |
Catalog# |
| KAPA Long Range DNA polymerase | 100 U | KK3006 |
| 250 U | KK3005 | |
| KAPA Long Range HotStart DNA polymerase | 100 U | KK3501 |
| 250 U | KK3502 | |
| 500 U | KK3503 | |
| KAPA Long Range HotStart Readymix with Dye | 100 rxn | KK3601 |
| 500 rxn | KK3602 |
Results
Amplification of 6 log dilution series of lambda DNA starting with 1 ng to 10 fg. Amplicons were 2 kb, 8 kb, and 15 kb in length. 35 cycles, 72ºC extension temperature, 1.25 units of enzyme.