| Conc.: 2.5 units/μL |
| Cat. No.: 210303 (500 Units) |
|
Cat. No.
|
Size Units
|
10X Ammonium Buffer
(MgCl2 15mM)
|
MgCl2
25 mM
|
|
210302
|
250
|
1.5 mL
|
1.5 mL
|
|
210303
|
500
|
1.5 mL
|
1.5 mL
|
|
210304
|
1000
|
2 x 1.5 mL
|
2 x 1.5 mL
|
|
210306
|
2500
|
4 x 1.5 mL
|
4 x 1.5 mL
|
|
| Store at -20°C. Reagent for in-vitro laboratory use only |
| |
| General Description |
| |
AccuPOL DNA Polymerase is a thermostable enzyme with proofreading ability, which can be used in primer extension reactions and other molecular biology appli-cations. AccuPOL exhibits both 5’→3’ DNA polymerase activity and 3’→5’ proofreading exonuclease activity. It is recommended for applications, which require extremely high fidelity or blunt ending.
Optimal reaction conditions are achieved by using the 10x Ammonium buffer containing MgCl2 provided with the enzyme. 25 mM MgCl2 is also included separately, in case a higher MgCl2 concentration is required for a specific reaction.
|
| |
|
| Key Features |
| |
‧Provides higher fidelity than Taq DNA Polymerase
‧Produces blunt-ended fragments
‧Processes <3 kb with extremely high fidelity |
| |
|
| Unit Definition |
| |
One unit is defined as the amount that incorporates 10 nmoles of dNTPs into acid-precipitable form in 30 minutes at 72°C under standard assay conditions. |
| |
|
| 10X Ammonium Reaction Buffer |
| |
Tris-HCl pH 8.5, (NH4)2SO4, 1% Tween® 20, 15mM MgCl2 |
| |
| AccuPOL Storage Buffer |
| |
50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, 0.1% NP40, 0.1% Tween-20. |
| |
|
| Suggested Protocol using AccuPOL DNA Polymerase |
| |
This protocol serves as a guideline for primer exten-sions. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be individually determined.
Set up reaction mixtures in an area separate from that used for DNA preparation or product analysis.
|
| |
|
- Thaw 10X Ammonium Buffer, dNTP mix, and primer solutions. It is important to mix the solutions completely before use to avoid localized concentrations of salts.
- Prepare a master mix according to Table 1. The master mix typically contains all the components needed for extension except the template DNA.
|
| |
|
| |
The optimal MgCl2 concentration should be determined empirically but in most cases a concentration of 1.5 mM, as provided in the 1X Ammonium Buffer, will produce satisfactory results. Table 2 provides the volume of 25 mM MgCl2 to add to the master mix if a higher MgCl2 concentration is required. |
| |
| |
Table 1. Reaction components (master mix and template DNA) |
| |
| Component |
Vol./reaction |
Final Conc. |
| 10X Ammonium Buffer |
5 μL |
1X |
| dNTP mix (12.5 mM of each) |
0.8 μL |
0.2 mM of each dNTP |
| Primer A |
Variable |
0.1–0.5 μM |
| Primer B |
Variable |
0.1–0.5 μM |
| AccuPOL DNA Polymerase |
1 μL |
2.5 units per reaction |
| Distilled Water |
Variable |
- - - - |
| Template DNA |
Variable |
0.1–0.5 μg per reaction |
| TOTAL volume |
50 μL |
- - - - |
|
| |
|
| |
Table 2. MgCl2 concentration in a 50μl reaction |
| |
| Final MgCl2 conc. in reaction (mM) |
1.5
|
2.0
|
2.5
|
3.0
|
3.5
|
4.0
|
4.5
|
| Additional volume of 25 mM MgCl2 per reaction (μL): |
0
|
1
|
2
|
3
|
4
|
5
|
6
|
|
| |
|
- Mix the master mix thoroughly and dispense appropriate volumes into reaction tubes. Mix gently, e.g., by pipetting the master mix up and down a few times.
- Add template DNA (0.1–0.5 μg/reaction) to the individual tubes containing the master mix.
- Program the thermal cycler according to the manufacturer’s instructions.
|
| |
|
| |
Notes: |
| |
- AccuPOL is a proofreading enzyme and require an extension time of 1-2 min./kb.
For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair. |
| |
|
- Place the tubes in the thermal cycler and start the reaction.
|
| |
|
| Quality Control |
| |
Endonuclease, exonuclease and priming activities are not detected after 3 hours incubation of 1 μg of pUC19 plasmid DNA and 0.5 μg EcoR I digested lambda phage DNA at 72°C in the presence of 40 units of AccuPOL DNA Polymerase.
|
| |
|
| Related Products |
| |
|
Description
|
Cat. No.
|
Taq DNA Polymerase (500 Units)
with 10X Ammonium Reaction Buffer
with 10X Standard Reaction Buffer |
110303
|
Taq DNA Polymerase (500 Units)
with 10X Combination Buffer |
110403
|
Taq DNA Polymerase (500 Units)
with 10X Mg++ Free Ammonium Buffer |
110503
|
Taq DNA Polymerase 2.0X Master Mix (100 Reac)
with 2.0 mM MgCl2 |
150301
|
Taq DNA Polymerase 2,0X MaMi RED (100 Reac)
with 1.5 mM MgCl2 |
180301
|
Taq DNA Polymerase 2.0X MaMi RED (100 Reac)
with 2.0 mM MgCl2 |
190301
|
| AccuPOL DNA Polymerase (500 Units) |
210303
|
TEMPase Hot Start DNA Polymerase (500Units)
with 10X TEMPase Buffer I
with 10X TEMPase Buffer II |
220303
|
| UniPOL –Long Range PCR (100 Reac) |
270701
|
| Rapid Ligation Kit (50 React) |
750300
|
| RT-PCR One Tube (100 Reac) |
740301
|
TEMPase Hot Start 2X Master Mix
with TEMPase Buffer I (100 Reac) |
230301
|
TEMPase Hot Start 2X Master Mix
with TEMPase Buffer II (100 Reac) |
230701
|
dNTP Mix (2 x 500μl)
(12.5 mM of each dA, dC, dG and dT) |
501004
|
dNTP Mix, (2 x 500 μl)
(10 mM of each dA, dC, dG and dT), |
502004
|
GC5 Value Efficiency, 108 Cfu/μg pUC19
Chemically Competent Cells, (10x 200μl) |
812010
|
GC5 High Efficiency, 109 Cfu/μg pUC19
Chemically Competent Cells, (10x 50μl) |
805010
|
GC5 High Efficiency, 109 Cfu/μg pUC19
Chemically Competent Cells, (5x 200μl) |
802005
|
SuperPath GC10, 1010 Cfu/μg pUC19
ElectroCompetent Cells, (5x 80μl) |
830805
|
| SOC Medium, 10x 10mL |
800000
|
|
| |
Tween 20® is a registered trademark of ICI Americas, Inc. |
| |
|
| |
NOTICE |
| |
In certain countries, patents cover the PCR process. This product is intended for researchers having a license to perform PCR or those not required to obtain a license.
|
| |
|
| |
|
