AUP-LJP- 250
A robust and very powerful Hi-fidelity polymerase meant for challenging PCR applications involving very difficult Genomic DNA templates of >3 kb to 18kb+ and Lambda DNA fragments of 28Kb+. The Product comes with a superior PCR Enhancer and Stabilizer to meet the toughest PCR situations
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(Suitable for both TA and blunt-end cloning)
SALIENT FEATURES AND BENEFITS-- The optimized composition of enzymatic activities enables AuPreP Longjump Polymerase to span the primer extension over long regions and demonstrate high processivity by reducing premature strand termination and template degradation. Using long primers at elevated Mg 2+ concentrations, >28Kb or >18Kb products can be achieved from lambda templates or genomic DNA, respectively. AuPreP Longjump Polymerase provides high performance and specificity, even with ‘dirty’ DNA or difficult templates with an unfavorable nucleotide composition. AuPreP Longjump Polymerase possesses 5'-3' DNA polymerase activity and 3'-5' proofreading activity which reduces mis-incorporations during PCR. This combination of properties provides much higher fidelity than Taq. In contrast with other proofreading enzymes, AuPreP Longjump Polymerase does not cause primers degradation
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Storage conditions : -20degrees Centigrade Conc: 4U/?l
Storage Buffer: 20mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1mM EDTA, 2mM DTT, 50% Glycerol & stabilizers
Endonuclease and Exonuclease activities: NIL Detected
NB: One unit is defined as the amount that incorporates 10nmoles of dNTPs into acid-precipitable form |
Each 250 units pack contains
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| AuPreP Longjump Polymerase…….. |
62.50 ?l |
| 10x Buffer |
1.2 ml |
| 50mM MgCl2 |
1.2 ml |
| DMSO |
1.0 ml |
| PCR Champ (enhancer) |
1.2 ml |
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IMPORTANT
Use of DMSO (supplied) in the reaction mix is recommended to increase the specificity of AuPreP Longjump Polymerase . Also supplied is a vial of PCR Champ (enhancer) , which helps to prevent the formation of false background bands and smearing, especially on difficult templates. PCR Champ (enhancer should be used at 1.0-2.0x final concentration - the optimal amount required should be determined for each individual experiment. PCR Champ (enhancer may also alter the ideal annealing temperature for primers - some optimization may be required. Please note that DMSO and PCR Champ (enhancer) \ should not be used together. |
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Last Updated on: 04/04/2009 AuPreP Longjump Polymerase Protocol & Usage Guidelines
Reaction Mix: |
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| AuPreP Longjump Polymerase 4u/?l |
-1?l |
| Template Lambda DNA 5ng/?l |
1?l |
| DMSO or PCR Champ (optional)+------ |
2.5?l |
| 10x Buffer |
5?l |
| 100mM dNTP |
0.5?l |
| Primer mix 100?M |
0.3?l |
| 50mM MgCl2 |
1?l |
| Water (ddH2O) |
Up to 50?l |
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| Cycling Parameters |
Stage of Incubation |
Incubation Temperature |
Incubation Time |
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| 1x |
Initial Denaturation |
94ºC |
2 min |
| 1x |
Annealing |
* |
1 min |
| 30x |
Denaturation |
94ºC |
30 sec |
| 30x |
Annealing |
* |
30 sec |
| 30x |
Extension |
68ºC |
10 min+ or 20++ min |
| 1x |
Final Elongation |
68ºC |
10 min+ or 20++ min |
| 1x |
Hold |
4ºC |
10 min+ or 20++ min |
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Annealing temperature is primer-dependent + 10 minutes for a 10 Kb fragment.
++20 minutes for a 20 Kb fragment
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This data is intended for use as a guide only; conditions will vary from reaction to reaction and may need optimization.
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PCR Troubleshooting Guide |
| If No or low PCR yield occurs, then the following can be the reasons along with the suggested solutions. |
- Enzyme concentration too low------------for this Increase the amount of enzyme in 0.5U increments.
- Magnesium concentration not optimized-------for this Increase concentration in 0.25mM increments.
- Primer concentration is optimized-------for this Titrate primer concentration (0.3-1:M);ensuring that both primers have the same concentration.
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| If Multiple bands occurs, then the following can be the reasons along with the suggested solutions. |
- Primer annealing temperature too low---------for this Increase annealing temperature. Primer annealing should be at least 5ºC below the calculated Tm of primers.
- Master mix left at room temperature------for this Prepare and keep master mix on ice
- Low Specificity --------- for this Add DMSO or PCR Champ as per suitability; please refer to page 1 of AuPreP Longjump Polymerase.
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| If Smearing or artefacts occur, then the following can be the reasons along with the suggested solutions. |
- Template concentration is too high --------- for this Prepare serial dilution of template.
- Too many cycles ---------- for this Reduce the cycle number by 3-5 to remove non-specific bands.
- Enzyme concentration is too high ------- for this Decrease the amount of enzyme in 0.5U increments.
- Extension time too long--------------- for this Reduce extension time in 0.5-1 minute increments.
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| Other AuPreP™ DNA/RNA Kits |
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