BEAMSBIO GS-Taq DNA Polymerase Set

GS-Taq DNA Polymerase (recombinant).  Store at –20℃
#3017S 1,000 U	2 vials of 500 U/vial : 5 U/µl. Supplied with 1.0 ml of 10X GS-Taq Rxn Buffer and  0.5ml of 10mM dNTP mix

Features

l         Taq DNA Polymerase fortified with High Fidelity and 3’à5’ proofreading activity

l         Generates TA cloning site of PCR products.

l         PCR for difficult genomic DNA and PCR above 10KB viral DNA template

l         RT-PCR

Description

GS-Taq DNA polymerase is heat stable Taq DNA polymerase optimized for  PCR above10KB. This enzyme has an optimum temperature around 74℃ and can withstand temperatures up to 95℃.  The enzyme has 3’à5’ proofreading activity with high fidelity. Our GS-Taq DNA polymerase is provided with x10 reaction buffer.  Our product has extremely high purity free from any nucleic acid contaminants and impurities,  which was purified through ion exchange column and affinity column chromatography.

Source

E. coli clone, recombinant gene from Thermus aquaticus YT1.

Concentration

5 U/µl.  Successful amplification can be achieved using 1~2U / 50㎕ reaction, typically for PCR target ~ 5 kb.  Further optimization, such as cycling time, reaction temperature, enzyme unit (2~4U/,50㎕), dNTP amount, and number of cycling, will be required for PCR target greater than 5~10kb.

Applications

l         PCR above 10KB viral DNA template and 5kb for genomic DNA

l         High throughput PCR and High reproducibility with 3’à5’ proofreading activity.

l         Generation of PCR products for TA cloning.

l         RT-PCR.

Quality Control

The BEAMSBIO GS-Tag DNA Polymerase is confirmed free of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases by strict quality tests, which shows satisfactory PCR results above 10 kb with viral DNA templates and up to 5 kb with genomic DNA templates.

Definition of Activity Unit

One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of dNTPs into an acid-insoluble product in 30 minutes at 74℃.

STANDARD UNIT ASSAY CONDITIONS 

50mM Tris-HCl (pH9.0 at 25℃), 50mM NaCl, 10mM MgCl₂, 200μM dATP, dCTP, dGTP, and radiolabeled dTTP, and 12.5μg activated calf thymus DNA in a 50μl reaction.

Storage Buffer

50mM Tris-HCl (pH8.0), 100mM NaCl, 0.1mM EDTA, 2mM DTT, 1.0% Triton X-100 and 50% glycerol.  Store at -20℃.

10X  Reaction Buffer

100mM Tris-HCl (pH8.3 at 25℃), 500mM KCl and 15mM MgCl₂. Buffer is optimized for use with 0.2mM for each of dNTPs.

Inhibition and Inactivation

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

BEAMSBIO GS-Taq DNA Polymerase Set 매뉴얼

PCR Protocol using GS-aq DNA Polymerase

1. Prepare PCR tubes for the experimental samples to be amplified.
증폭할 실험시료에 적합한 PCR튜브를 준비한다.

2. Add the components in order according to following recipe :
다음 성분표를 참조하여 실험용 재료를 순서대로 넣는다.

  Distilled water,                                                      36~43㎕ * (balance to total vol. 50㎕)
  x10 Reaction buffer,                        5㎕
  10mM dNTP mix,                                              1~3㎕
  GS-Taq DNA Polymerase,                                    0.5~1㎕ (5U/㎕) (투입 후 가볍게 섞는다)

  DNA templates(10~200ng/㎕),                     0.2~1㎕
  Primer, forward(10pmol/㎕),                             0.2~2㎕
  Primer, backward(10pmol/㎕),                           0.2~2㎕
-----------------------------------------------------------------------------
                                                                 total  50㎕
 * Primers(template와 미리 배합 사용가능)를 마지막에 넣는 것이 좋다.

3. Mix gently and rapidly. Centrifuge down PCR tubes immediately after mixing.
가볍고 신속하게 섞고, 즉시 짧게 원심분리하여 반응물을 아래로 모은다.

4. Perform PCR using optimized cycling conditions.  Suggested cycling parameters for PCR using PCR Master Mix are :
다음 운전조건을 참조하여 PCR Master Mix 최적 PCR운전조건을 설정하여 실험을 수행한다.

  - Step 1 :  initial denaturation,                                     94~95℃ for 45sec.
  - Step 2 :  denaturation,                                    94℃ for 45sec.
            annealing,                                    Tm-5℃ for 45sec.
            extension,                                    68~72℃ for 0.5~1 min/kb
           * 25~30 cycles recommended
           * 10KB 내외인 경우, 10~15 싸이클 수행후 20~30초씩 시간을
              늘리면서 15~20 싸이클 추가로 수행함(조건 최적화 필요).
  - Step 3 :  final extension,                                    72℃ for 7~20min.

5. Cool down below 15℃ until harvest.

PCR Troubleshooting

1. PCR band가 전혀 없는 경우 :
- 반응물 성분 배합에 착오가 없는지 확인한다.  성분누락 여부 ?
- PCR 운전조건이 적합하였는지 확인한다.  PCR반응기 운전 실수 여부 ?
- PCR Master Mix용도에 맞지 않는 DNA template 증폭시도 여부 ?
 Long PCR Master Mix, Pfu PCR Master Mix 등 적합한 PCR System 선택
- Primer Design 적합, Template DNA 손상, Inhibitor 잔존 여부 등 검토

2. PCR band가 뿌옇게 ㈜빔스바이오에서 판매한 바이오소재 제품의 품질 불만에 대하여 공급자를 통하여 당사와 미리 협의가 완료된 경우 별도의 비용 부담없이 제품을 교환하여 드립니다. 따라서, 품질 불만에 대하여 미리 협의가 이루어지기 전까지는 물품을 임의로 반송하여서는 안되며, 이 경우 발생하는 모든 비용은 고객이 부담하여야 합니다. 바이오 소재 제품은 운송 시 품질이 변동될 소지가 크기 때문에 제품 품질 문제는 대개 운송 도중에 발생합니다.  당사에서는 반송된 물품을 검사 후 검사 결과에 상관없이 즉시 폐기합니다.

A return authorization is required before shipping.  Please do not return any product without first contacting Beams Biotechnology or distributor for a return authorization.  All return authorizations must be issued directly from Beams Biotechnology.  In the case of a purchasing error, it is our policy to deny return of product.  Due to the temperature sensitivity of the products, we are unable to resell returned goods, as we are not assured of the quality.