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Pfu DNA Polymerase Set(dNTP mix Æ÷ÇÔ), High Fidelity, cat.#3016S/500U
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BEAMSBIO Pfu DNA Polymerase Set

Pfu DNA Polymerase (recombinant).  Store at –20¡É

#3016S
500 U

2 vials of 250 U/vial : 2.5 U/µl. Supplied with 1.0ml of 10X Pfu Rxn Buffer with MgSO4 and 0.5ml of 10mM dNTP mix


Features

l      Eight times more accurate than Taq DNA polymerase.

l      Highly thermostable - remains 95% active after 2 hours incubation at 95¡ÆC.

l      Generates blunt-end PCR products.

l      Incorporates modified nucleotides (e.g., biotin-, digoxigenin-, fluorescently-
   labeled nucleotides).

Description

Pfu DNA Polymerase, a proofreading DNA polymerase, is a highly thermostable DNA polymerase originated from the hyperthermophilic archaeum Pyrococcus furiosus, retaining 94-99% of its polymerase activity after 1 hour at 95¡ÆC. To amplify GC-rich regions, denaturation temperature up to 98C can be used to with Pfu DNA polymerase.. The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5'à3' direction. The Pfu DNA Polymerase exhibits 3'à5' exonuclease (proofreading) activity only which enables the polymerase to correct nucleotide incorporation errors, but no 5'à3' exonuclease activity.  Successful PCR using Pfu DNA polymerase is readily performed requiring slight modifications from PCR protocols optimized with Taq DNA polymerase.

Source

Pfu DNA Polymerase (recombinant) : E.coli cells with a cloned gene originated from Pyrococcus furiosus.  Molecular Weight of cloned Pfu DNA Polymerase (recombinant) is 90 kDa.

Concentration

2.5 U/µl.  Successful amplification can be achieved using 1~2.5U / 50§¡ reaction, typically for PCR target < 2kb.  Further optimization, such as cycling time, reaction temperature, enzyme unit, dNTP amount, and number of cycling, will be required for PCR target greater than 2kb.

Applications

l      High fidelity PCR

l      Blunt-end PCR cloning

l      Site-directed mutagenesis.

Quality Control

The BEAMSBIO Pfu DNA Polymerase is confirmed free of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases by strict quality tests, which shows satisfactory PCR results upto 10 kb with viral DNA templates and up to 4 kb with genomic DNA templates.

Definition of Activity Unit

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 72¡ÆC.  Enzyme activity is assayed in the following mixture: 20 mM Tris-HCl (pH 8.8 at 25¡ÆC), 2 mM MgSO4, 10 mM (NH4)2SO4, 10 mM KCl, 0.1% (v/v) Triton X-100, 0.1 mg/ml BSA, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [3H]-dTTP.

Storage Buffer

The enzyme is supplied in: 50 mM Tris-HCl (pH 8.2), 1 mM DTT, 0.1 mM EDTA, 0.1% (v/v) Nonidet P40, 0.1% (v/v) Tween 20 and 50% (v/v) glycerol.

10X Pfu Reaction Buffer

200 mM Tris-HCl (pH 8.8 at 25¡ÆC), 100 mM (NH4)2SO4, 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO4.

Inhibition and Inactivation

Inactivated by phenol/chloroform extraction.  Heat denaturation is not possible.

 

PCR Protocol using Pfu DNA Polymerase

1. Prepare PCR tubes for the experimental samples to be amplified.
ÁõÆøÇÒ ½ÇÇè½Ã·á¿¡ ÀûÇÕÇÑ PCRÆ©ºê¸¦ ÁغñÇÑ´Ù.

2. Add the components in order according to following recipe :
´ÙÀ½ ¼ººÐÇ¥¸¦ ÂüÁ¶ÇÏ¿© ½ÇÇè¿ë Àç·á¸¦ ¼ø¼­´ë·Î ³Ö´Â´Ù
.

  Distilled water,                                   31~40
§¡ * (balance to total vol. 50§¡)
  x10 Reaction buffer,                                   5
§¡

  10mM dNTP mix,                                   1~3
§¡
  Pfu DNA Polymerase,                               1
§¡ (2.5U/§¡) (ÅõÀÔ ÈÄ °¡º±°Ô ¼¯´Â´Ù)
  DNA templates(10~200ng/§¡),          0.2~1 §¡
  Primer, forward(10pmol/
§¡),              0.2~2 §¡
  Primer, backward(10pmol/
§¡),          0.2~2 §¡
-----------------------------------------------------------------------------
                                                         total     50
§¡
 * Primers(template
¿Í ¹Ì¸® ¹èÇÕ »ç¿ë°¡´É)¸¦ ¸¶Áö¸·¿¡ ³Ö´Â °ÍÀÌ ÁÁ´Ù.
 * Magic buffer,   2~5 §¡ (ÇÊ¿ä ½Ã¿¡¸¸ Àû·® »ç¿ë)

3. Mix gently and rapidly. Centrifuge down PCR tubes immediately after mixing.
°¡º±°í ½Å¼ÓÇÏ°Ô ¼¯°í, Áï½Ã ª°Ô ¿ø½ÉºÐ¸®ÇÏ¿© ¹ÝÀÀ¹°À» ¾Æ·¡·Î ¸ðÀº´Ù.

4. Perform PCR using optimized cycling conditions.  Suggested cycling parameters for PCR using PCR Master Mix are :
´ÙÀ½ ¿îÀüÁ¶°ÇÀ» ÂüÁ¶ÇÏ¿© PCR Master Mix ÃÖÀû PCR¿îÀüÁ¶°ÇÀ» ¼³Á¤ÇÏ¿© ½ÇÇèÀ» ¼öÇàÇÑ´Ù
.
  - Step 1 :  initial denaturation,               94~95¡É
for 2~5 min.
  - Step 2 :  denaturation,                         94
¡É
for 45 sec.
                   annealing,                              Tm-5
¡É
for 45 sec.
                   extension,                              68~74
¡É
for 1~2 min/kb
                   * 25~30 cycles recommended, upto 40 cycles possible
  - Step 3 :  final extension,                      72
¡É for 5~10 min.

5. Cool down below 15¡É until harvest.

PCR Troubleshooting

1. PCR band°¡ ÀüÇô ¾ø´Â °æ¿ì :
- ¹ÝÀÀ¹° ¼ººÐ ¹èÇÕ¿¡ Âø¿À°¡ ¾ø´ÂÁö È®ÀÎÇÑ´Ù.  ¼ººÐ´©¶ô ¿©ºÎ
?
- PCR
¿îÀüÁ¶°ÇÀÌ ÀûÇÕÇÏ¿´´ÂÁö È®ÀÎÇÑ´Ù.  PCR¹ÝÀÀ±â ¿îÀü ½Ç¼ö ¿©ºÎ
?
- PCR Master Mix
¿ëµµ¿¡ ¸ÂÁö ¾Ê´Â DNA template ÁõÆø½Ãµµ ¿©ºÎ
?
 Long PCR Master Mix, Taq PCR Master Mix
µî ÀûÇÕÇÑ PCR System ¼±ÅÃ

- Primers Design
ÀûÇÕ ¿©ºÎ?

2. PCR band°¡ »Ñ¿¸°Ô ²ø¸± ¶§ :
- Target bandº¸´Ù ¾Æ·¡ÂÊ¿¡¼­ ÀÛÀº non-specific band°¡ ¸¹ÀÌ °üÂûµÉ ¶§´Â
,
             . primer design
À» Àç°ËÅäÇÑ´Ù
.
             . DNA template
ºÒ¼ø¹°ÀÌ ³ôÀ¸¸é ¼øµµ¸¦ ³ôÀδÙ
.
             . annealing temp.
¸¦ 1~2¡É ³ô¿© º»´Ù
.
             . extension
¿Âµµ¸¦ Á¶±Ý ³ôÀ̰ųª ¹ÝÀÀ½Ã°£À» Á¶±Ý ´Ã¸°´Ù

             .
±æÀÌ°¡ ±æ°Å³ª ÁõÆøÀÌ ¾î·Á¿î DNA templateÀ̶ó¸é Long PCR
               Master Mix
·Î º¯°æÇÏ¿© ´Ù½Ã ½ÃµµÇÑ´Ù
.
- Target band
º¸´Ù ÀüüÀûÀ¸·Î ²ø¸± ¶§´Â
,
             . PCR
¹ÝÀÀ¿Âµµ¸¦ Á¶Á¤ÇÑ´Ù. Annealing ¿Âµµ¸¦ Tm-5¡É ºÎ±Ù¿¡¼­

              1~2
¡É ³·Ãá´Ù.
             . extension
¿Âµµ¸¦ 68~72¡É ³»¿¡¼­ ³·Ãß°í,¹ÝÀÀ½Ã°£À» Á» ÁÙÀδÙ
.
              DNA template
±æÀÌ°¡ 1kbÀÌ»óÀ̸é 0.5~1 min/kb ¹üÀ§¿¡¼­ ´Ù¼Ò

             
ÁÙÀÌ°í, 20 cycleÀÌÈÄ¿¡¼­ ¸Å cycle´ç 10~20sec.¾¿ ´Ã¸°´Ù.
- PCR target band
´Â Àß ³ª¿À³ª ²ø¸² Çö»óÀÌ ½ÉÇÒ ¶§,
             . DNA template and/or Primers »ç¿ë·®À» Á» ÁÙÀδÙ.
... ÀÌÇÏ »ý·«.  Á¦Ç° ¸Å´º¾ó ÂüÁ¶ ...


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