A Simple Solution for Oligonucleotide Design

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Oligo Sequence:

Tm:
°C± °C
Oligo length:
bp± bp



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Common Data ID:

Taxonomy ID:

GI Number:

CommonData ID: BM1806Publication date: 12/01
PMID: 11878878GI: 2465191 Taxonomy ID: 11099
Virus Name: pestivirus type 1Product size (PCR):
bp
MgCl2: 2.5 mMdNTPs: mMType: RT-Nested PCR 1(1)
Temp (PCR cycle):(94C 30sec>55C 30sec>72C 60sec)*35>68C 10min
Buffer: 25mM Tris-HCl (pH 8.3), 50mM KCl, 0.1% Triton X-100, 0.02% BSA
Polymerase: 0.1U/microL Taq poly (QBiogene)Cycler:
Note: Primer sets Ol-B32II/Ol-JasIns1, Ol-JasIns2/Ol-B22.1R, and Ol-BVDV32/Ol-JasIns1 are used in separate reactions.
Primer set Ol-BVDV32/Ol-JasIns1 detected pestiviruses with internal deletions most effectively.

11.5microL RNA-primer mixture containing 173.9ng/microL RNA and 2.61microM reverse primer are incubated 94C 3min>8C 10min.
RT is performed in 20microL total volume containing 11.5microL RNA-primer mixture, 50mM Tris-HCl (pH 8.3), 73mM KCl, 3.0mM MgCl2, 10mM DTT, 0.5mM each dNTP, 0.75U/microL RNAguard (Pharmacia), and 2.5U/microL Superscript RTase (Life Technologies BRL).
RT temperature: 37C 45min

PCR is performed in 50microL containing all 20microL cDNA and a primer set.
Article does not mention concentration of forward primer used in PCR.

For secondary PCR, [see BM1811].
For other primary PCRs, [see BM1807], [see BM1808], [see BM1809], and [see BM1810].
Reverse(Ol-B22.1R) primer
Oligo type: PCRDegeneracy: 1
Target: 2198-2178Tm: 70.5 C at salt 1000.0mMLength:
Sequence: GTTGACATGGCATTTTTCGTG
(4 other experimental conditions)
Note: Mismatches at positions 2198 and 2192.
Forward(Ol-B32II) primer
Oligo type: Degeneracy: 1
Target: 1163-1183Tm: 78.3 C at salt 1000.0mMLength:
Sequence: CCAAGTCCCTGTGCTGTTCCC
(1 other experimental conditions)
Note:
Reverse(Ol-JasIns1) primer
Oligo type: Degeneracy: 1
Target: 1882-1865Tm: 54 CLength:
Sequence: GGGATTTTGGTAGGATGC
(1 other experimental conditions)
Note:
Forward(Ol-JasIns2) primer
Oligo type: Degeneracy: 1
Target: 1836-1852Tm: 50 CLength:
Sequence: GTAGAAGATGTCCGACT
(2 other experimental conditions)
Note:
Forward(Ol-BVDV32) primer
Oligo type: Degeneracy: 1
Target: 347-371Tm: 75.5 C at salt 1000.0mMLength:
Sequence: AAATCTCTGCTGTACATGGCACATG
(1 other experimental conditions)
Note: Specific to [GI:1518835].
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