- Thermolase-Thermostable DNA polymerase.
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- Description
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- Thermolase (EC 2.7.7.7.) is a thermostable DNA-dependent DNA
polymerase isolated from Thermus sp. and now available in a genetically
engineered form. The enzyme has 5´-3´ polymerase (60-150
nt/s, about 1 kb/min), 5´-3´ exonuclease (strand displacement),
3´ terminal deoxynucleotidyl transferase actifity (which usualy
results in addition of single dATP to the duplex DNA). Modified nucleotides
(dNTPaS, c7GTP, biotin-11-dUTP, digoxigenin-11-dUTP and fluorescein-12-dUTP
but not biotin-16-dUTP) are incorporated at high rates by the enzyme.
Error rates are 1x10-4 for misincorporation and 2x10-5
for frameshift mutation. Only the last three bases adjacent to the 3´
end of the primer need to be correctly base paired, in order to initiate
polymerization. The 5´ region of the primer is less sensitive
to base mismatches. Therefore, new restriction sites can be easily introduced
into an amplification product. Up to 9 kb can be amplified from lambda
DNA and up to 5 kb from genomic DNA.
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- Unit definition
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One unit of the enzyme will incorporate 10 nmol of dNTPs into an acid-insoluble
form in 30 min at 72°C under standard assay conditions using a DNA
template. Specific activity is 200.000U/mg protein.
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- Reaction buffers
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B1 (x10, with Mg2+ ): 670 mM Tris-HCl,
pH 8.8, 166 mM (NH4)2SO4, 15
mM MgCl2, 0.1% Tween 20. |
- Reaction conditions
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- For sample volume 20-100 µl use 200 mM dNTPs (end conc.), 25
pM (abs. quantity) each primer, 2 U of the enzyme, 100 ng genomic DNA
as template. Addition of excess enzyme or template may lead to nonspecific
amplification. For fragments <1 kb, please dilute the enzyme 1:5
with 1x buffer just before use.
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Amplification conditions
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For amplification of 3kb fragment please use 0,2 ml tubes and following
conditions:
10x reaction buffer
2 mM dNTPs (200 µM each)
human genomic DNA (100 ng)
forward primer factor VIII.1(25 pM)
reverse primer factor VIII.4 (25 pM)
Thermolase (5 units/µl)
H2O
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3 µl
3 µl
1 µl
1 µl
1 µl
0,5 µl
20,5 µl
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total
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30 µl
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Hot-start
Steps
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- 94° 3 min
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- 58° 0,5 min
- 72° 4 min
- 93° 15 sec
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Number of cycles
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30 cycles
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- Storage
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- Store the control DNA at +4°C to avoid degradation by multiple
freezing and thawing. Store all other reagents at -20°C.
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- Applications
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- PCR, mutation analysis (TaqMan, PAMSA), site-directed mutagenesis,
DNA labeling, 3´ A-tailing of blunt ends; primer extension; sequencing.
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Quality control
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activity, SDS-PAGE purity, absence of endo- and exonucleases, specific
performance tests.
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References
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1. Chien, A. et al. (1976) J. Bacteriol. 127, 1550. 2. Kaledin, A.S.
et al. (1980) Biokhimiya 45, 494. 3. Clark, J.M. (1988) Nucleic Acids
Res. 18, 9677-9686.
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- Catalog #
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- T1.4
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T1.5
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Pack size
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1000 U
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2500 U
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