UniPol-Long Range PCR
Code  BN0110-0100
Classification  PCR試劑 & 1-Step Kit
Size  100R
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UniPOL For Long Range PCR

With 10X UniPOL Buffer A (MgCl2 15mM)
With 10X UniPOL Buffer B (MgCl2 15mM)
Cat. No.: 270701 (100 Reactions)
Cat. No.
Size Units
10X UniPOL Buffer A
(MgCl2 15mM)
10X UniPOL Buffer B
(MgCl2 15mM)
DMSO
270701
100
1.5 mL
1.5 mL
0.5 mL
270702
200
1.5 mL
1.5 mL
0.5 mL
270703
500
1.5 mL
1.5 mL
1.5 mL
270704
1000
2 x 1.5 mL
2 x 1.5 mL
1.5 mL
270706
2500
4 x 1.5 mL
4 x 1.5 mL
1.5 mL
Store at -20°C. Reagent for in-vitro laboratory use only
 
General Description
 

Ampliqon UniPOL-Long Range PCR Enzyme mix, combines Ampliqon’s high quality Taq DNA Polymerase with a small amount of AccuPOL, which exhibit 3’→5’ proofreading exonuclease activity. The UniPOL Enzyme mix is optimised for amplifications of 5-20 kb with a high yield.

Amplification using Taq DNA Polymerase is generally limited to amplifying up to 4-6 kb, depending on template. This is partly due to the lack of 3’→5’ proofreading exonuclease activity of the Taq DNA Polymerase. Mis-incorporation of nucleotides often leads to processive mistakes and consequently a terminal event will occur and the elongation will arrest. A mix of the processive Taq DNA Polymerase and the AccuPOL proofreading enzyme, which corrects mis-incorporated nucleotides, increases the length of the amplification product.

Due to differences in complexity of template, Ampliqon has optimized two different Buffers- UniPOL Buffer A and UniPOL Buffer B.

Some difficult templates require additives, such as DMSO, which has been reported to give DNA thermal stability against depurination and to be useful for G-C rich templates.
   
10X UniPOL Buffer A
  Tris-HCl pH 8.9, KOAC, 15 mM MgCl2,
   
10X UniPOL Buffer B
  Tris-HCl pH 8.9, KOAc, 15mM MgCl2, and Stabilizer
   
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.
   
Storage and Dilution Buffer
 
Enzyme is supplied in 20 mM Tris-HCl pH 8.3, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20®, 0.5% NP40, 50% glycerol.
   
Quality Control
 
Each lot of UniPOL is tested for contaminating activities, with no trace of endonuclease activity, nicking activity, exonuclease activity or priming activity.
 
Suggested Protocol using UniPOL-Long Range PCR
 
This protocol serves as a guideline for primer extensions. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be individually determined.
   
  Notes:
 
  1. Set up reaction mixtures in an area separate from that used for DNA preparation or product analysis.
  2. 15mM MgCl2 is present in the 10X buffers. The 1X concentration is 1.5mM MgCl2.
  3. Addition of DMSO to a final concentration of 1-5% may increase yield and improve reliability of the system for some complex PCR targets.
  4. Effective denaturation can be accomplished by the use of higher temperature for shorter periods of time or by adding DMSO.
  5. Reliable amplification of long DNA sequences requires:
    1. Effective denaturation of DNA template
    2. Protection from depurination.
    3. Extension time long enough to produce large products.
    ( a. and b. can be achieved with addition of DMSO, if necessary).
   
  1. Thaw 10x UniPOL Buffer A and/or 10x UniPOL Buffer B, dNTP mix, primer solutions. It is important to mix the solutions completely before use to avoid localized concentrations of salts.
  2. Prepare a master mix according to Table 1. The master mix typically contains all the components needed for extension except the template DNA.
   
  Table 1. Reaction components (master mix and template DNA)
 
Component Vol./reaction Final Conc.
10X UniPOL Buffer A or
10x UniPOL Buffer B		 5 μL 1X
dNTP mix
(12.5 mM of each)		 0.8 μL 0.2 mM of each dNTP
Primer A Variable 0.1–1.0 μM
Primer B Variable 0.1–1.0 μM
UniPOL
Long Range PCR		 0.5μL  
Distilled Water Variable - - - -
Template DNA Variable Variable
Optional:
DMSO (100%)		 0.5μL – 2.5 μL 1 – 5%
TOTAL volume 50 μL - - - -
   
  Table 2. MgCl2 concentration in a 50 μL 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
   
  1. 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.
  2. Add template DNA to the individual tubes containing the master mix.
  3. Program the thermal cycler according to the manufacturer’s instructions.

    For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.

    Both two and three step PCR can be used;
   
  Ex. Three step PCR
  25-40 cycles:
 
┌ 95°C 10-30 sec. Denature template
│ 55-68°C 10-30 sec. Anneal primer
└ 72°C 1-20 min. Elongation*
  72°C for 10 min. Final Elongation
  *Recommended time is approx. 45 sec. per kb of target
   
  Ex. Two step PCR
  25-40 cycles:
 
  95°C 10-30 sec. Denature template
  68°C 10-30 sec. Anneal primer & Elongation *
  72°C for 10 min. Final Elongation
  *Recommended time is approx. 45 sec. per kb of target
   
  1. Place the tubes in the thermal cycler and start the reaction.
   
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805010
GC5 High Efficiency, 109 Cfu/μg pUC19
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  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.