Kit for detecting polynucleotide kinase (PNK) and detection method for kit
A polynucleotide kinase and detection method technology, which is applied in the detection kit of polynucleotide kinase and its detection field, can solve the problems of incomplete fluorescence quenching effect, insufficient support for practical application, lack of signal amplification strategy, etc., and achieve Fast and efficient detection, reduced experimental cost, and low detection limit
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Embodiment 1
[0043] Embodiment 1 A kind of test kit for detecting polynucleotide kinase
[0044] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.
[0045] Said mixed liquid A comprises: 0.05 milligram per milliliter of bovine serum albumin, 0.1 unit per microliter of Nt.BbvCI nicking enzyme, 0.05 unit per microliter of KF polymerase, 200 micromoles per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.
[0046] The nucleotide sequence of the probe A is 5'-GCA CAA AGG ACT GAG GCT GAG GGT TGC GCA TCTGCT ATG AAA CAA GCC AGA TGC TCA AC-3', as shown in SEQ ID NO.1, its 5' end Phosphorylation modification; the nucleotide sequence of probe S is 5'-GTT GAG CAT CTG GCT TGT TTC ATA GCA GAT GTT TTT T-3', as shown in SEQ ID NO.2, and its 5' end is phosphorylated grooming.
[0047] The template D...
Embodiment 2
[0049] Embodiment 2 A kind of test kit for detecting polynucleotide kinase
[0050] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.
[0051] Said mixed liquid A comprises: 0.15 milligrams per milliliter of bovine serum albumin, 0.3 units per microliter of Nt.BbvCI nicking enzyme, 0.15 units per microliter of KF polymerase, 300 micromoles per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.
[0052] Said mixed liquid A comprises: 0.1 milligram per milliliter of bovine serum albumin, 0.2 unit per microliter of Nt.BbvCI nicking enzyme, 0.1 unit per microliter of KF polymerase, 250 micromole per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.
[0053] The nucleotide sequences of the probe A, probe S and template DNA are similar to Example 1.
[005...
Embodiment 3
[0055] Embodiment 3 A kind of test kit for detecting polynucleotide kinase
[0056] The kit for detecting polynucleotide kinase comprises the following components: probe A, probe S, mixed solution A, template DNA, lambda exonuclease, polynucleotide kinase standard product, 1×NEBuffer and 10 mM adenosine triphosphate.
[0057] Said mixed liquid A comprises: 0.1 milligram per milliliter of bovine serum albumin, 0.2 unit per microliter of Nt.BbvCI nicking enzyme, 0.1 unit per microliter of KF polymerase, 250 micromole per liter of deoxynuclear triphosphate nucleotides and 10X SYBRgreen I dye.
[0058] The nucleotide sequences of the probe A, probe S and template DNA are similar to Example 1.
[0059] The composition and concentration contained in the 1×NEBuffer are similar to those in Example 1.
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