PDF《M0493S》

Q5? Hot Start High-Fidelity DNA Polymerase M0493S

100 units 2,000 U/ml Lot:

0011207 RECOMBINANT Store at C20°C Exp: 7/14 Reagents Supplied with Enzyme: 5X Q5 Reaction Buffer 5X Q5 High GC Enhancer Reaction Conditions: 1X Q5 Reaction Buffer, DNA template, 0.

5 μM primers,

200 μM dNTPs (not included), 1X Q5 High GC Enhancer (optional) and

1 unit of Q5 Hot Start High-Fidelity DNA Polymerase in a total reaction volume of

50 μl. Unit Definition: One unit is defined as the amount of enzyme that will incorporate

10 nmol of dNTP into acid insoluble material in

30 minutes at 74°C. Unit Assay Conditions:

25 mM TAPS-HCl (pH 9.3 @ 25°C),

50 mM KCl,

2 mM MgCl2 , 1?mM?β-mercaptoethanol,

200 μM dNTPs including [3 H]-dTTP and

400 μg/ml activated Calf Thymus DNA. Heat Inactivation: No (see other side) Enhancer-Dependent High GC (65% GC) PCR: 30?cycles of PCR amplification in a

50 μl reaction containing

20 ng genomic DNA with 1.0 unit of Q5 Hot Start High-Fidelity DNA Polymerase in the presence of

200 μM dNTPs, 1X Q5 High GC Enhancer and 0.5 μM of each primer in Q5 Reaction Buffer result in the enhancer-dependent production of the

452 bp high GC product. Hot Start-Specific Genomic DNA PCR:

25 cycles of PCR amplification in a

25 μl reaction contain- ing 50?ng genomic DNA with 0.5 units of Q5 Hot Start High-Fidelity DNA Polymerase in the presence of 200?μM dNTPs, 1X Q5 High GC Enhancer and 0.5?μM of each primer in Q5 Reaction Buffer result in the expected

665 bp product, free of non-specific amplification products after pre-incubation at room temperature for

1 hour. Endonuclease Activity: Incubation of a

50 μl reaction in NEBuffer

2 containing a minimum of 10?units of Q5 High-Fidelity DNA Polymerase with

200 μM dNTPs and

1 μg of supercoiled pUC19 DNA for

4 hours at either 37°C or 72°C results in