[NIPPON GENE] Thermostable Strand-Displacement DNA Polymerase

Product name	Optimal temperature	Deactivation temperature*1	Component
BST DNA Polymerase	60～65℃	80℃, 5 min	1) BST DNA Polymerase (8units/μl) 2) 10× BST Reaction Buffer (80mmol/l Mg2+)	1,600units 500μl
Csa DNA Polymerase	60～70℃	85℃, 5 min	1) Csa DNA Polymerase (8units/μl) 2) 10× Csa DNA Polymerase (80mmol/l Mg2+)	1,600units 500μl
96-7 DNA Polymerase	50～55℃	70℃, 5 min	1) 96-7 DNA Polymerase (8units/μl) 2) 10× 96-7 DNA Polymerase (95mmol/l Mg2+)	1,600units 500μl

*1 Inactivation temperature when enzyme stock solution is directly heat denatured.

LAMP reaction conditions

• Bst DNA Polymerase
  Reaction: 65°C, 60 min

  <Composition of reaction mixture>

  Candidatus Liberibacter asiaticus DNA※2	1×106copies
  FIP	40pmol
  BIP	40pmol
  F3 Primer	5pmol
  B3 Primer	5pmol
  Loop Primer F	20pmol
  Loop Primer B	20pmol
  dNTPs Mixture	1.4mM each
  10× Bst Reaction Buffer	2.5 μl
  Bst DNA Polymerase	8units
  Total	25μl

  *2 The LAMP primer set for detection of Candidatus Liberibacter asiaticus was developed by the Kyushu Okinawa Agricultural Research Center of the National Agriculture and Food Research Organization under a project titled "Development of technology to prevent the spread of pesticide-resistant citrus greening disease," one of their projects promoting agricultural forestry and fisheries research using advanced technology.

• 

  ＜Lane＞

  M：Gene-Ladder Wide 1(Product Number 313-06961)
  1 : Company A Bst DNA Polymerase
  2 : NIPPONGENE Bst DNA Polymerase

  ＜Note＞

  3.0% agarose 21/TAE gel electrophoresis
  Ethidium bromide staining

Primer sequence

FIP: 5'-GCATGCCGAGGATCAATGCCTTGCTTAAAGAGCGTGCTACG-3'
BIP: 5'-TATGCCTAATGGCACGGGGGTAAGCTTCATCCGCCTTCGA-3‘
F3 Primer: 5'-TGGGTTAAGTGATGCTGTGG-3'
B3 Primer: 5'-CAACAATATCAGCCCCTGCT-3'
Loop Primer F: 5'-TCTCAACTGTTTCATCAAACCTAGC-3'
Loop Primer B: 5'- CGTGGCGGTTTTTGCTACA-3'

LAMP reaction conditions

<Composition of reaction mixture>

• Csa DNA Polymerase

  Candidatus Liberibacter asiaticus DNA	1×106copies
  FIP	40pmol
  BIP	40pmol
  F3 Primer	5pmol
  B3 Primer	5pmol
  Loop Primer F	20pmol
  Loop Primer B	20pmol
  dNTPs Mixture	1.4mM each
  10× Csa Reaction Buffer	1×
  Csa DNA Polymerase	8units
  total	25μl

  Reaction: 65°C, 60 min

• 96-7 DNA Polymerase

  Candidatus Liberibacter asiaticus DNA	1×106copies
  FIP	40pmol
  BIP	40pmol
  F3 Primer	5pmol
  B3 Primer	5pmol
  Loop Primer F	20pmol
  Loop Primer B	20pmol
  dNTPs Mixture	2.0mM each
  10× 96-7 Reaction Buffer	1×
  96-7 DNA Polymerase	8units
  total	25μl

  Reaction: 65°C, 60 min

Primer Sequence

• FIP: 5'-GCATGCCGAGGATCAATGCCTTGCTTAAAGAGCGTGCTACG-3'
  BIP: 5'-TATGCCTAATGGCACGGGGGTAAGCTTCATCCGCCTTCGA-3‘
  F3 Primer: 5'-TGGGTTAAGTGATGCTGTGG-3'
  B3 Primer: 5'-CAACAATATCAGCCCCTGCT-3'
  Loop Primer F: 5'-TCTCAACTGTTTCATCAAACCTAGC-3'
  Loop Primer B: 5'- CGTGGCGGTTTTTGCTACA-3'

• <Note>

  3.0% agarose 21/TAE gel electrophoresis
  Ethidium bromide staining

• 

• <Lane>

  M： Gene-Ladder Wide 1(Product Number 313-06961)
  1： Strand-displacement DNA polymerase derived from Geobacillus
  2： NIPPONGENE Csa DNA Polymerase
  3： NIPPONGENE 96-7 DNA Polymerase

RCA reaction conditions

<Composition of reaction mixture>

• M13mp18 single strand DNA	20ng
  Universal Primer	50nM
  dNTPs Mixture	0.25mM< each
  Tris-HCl (pH 8.8 at 25℃）	20mM
  KCl	10mM
  (NH4)2SO4	10mM
  MgSO4	2mM
  Tween 20	0.1%
  DNA Polymerase	8units
  total	20μl

• 

  ＜Lane＞

  M： Gene-Ladder Wide 1(Product Number 313-06961)
  D： Template DNA(M13mp18) single strand DNA

Primer Sequence

Universal Primer
5'-GTTTTCCCAGTCACGACGTTGTA-3'

<Note>

3.0% agarose 21/TAE gel electrophoresis
Ethidium bromide staining

RCA(Rolling Circle Amplification) reaction diagram