Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.

cgi

Primer-BLAST Primer-Blast results

NCBI/ Primer-BLAST : results: Job id=LCbzRbPJvmGZWy5eIz4KbFklG150NgBDdQ more...

Input PCR template

AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene, complete cds;
tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial products
Range
1 - 1156
Specificity of primers
Primer pairs are specific to input template as no other targets were found in selected database: Custom (Organism limited to Draco spilopterus)
Other reports
Search Summary

Search parameters and other details

Search parameter name Search parameter value
Number of Blast hits analyzed 64
Entrez query
Min total mismatches 2
Min 3' end mismatches 2
Defined 3' end region length 5
Mismatch threshold to ignore targets 6
Max target size 4000
Max number of Blast target sequences 50000
Blast E value 30000
Blast word size 7
Max candidate primer pairs 50
Min PCR product size 70
Max PCR product size 1000
Min Primer size 15
Opt Primer size 20
Max Primer size 25
Min Tm 57
Opt Tm 60
Max Tm 63
Max Tm difference 3
Repeat filter AUTO
Low complexity filter Yes

1 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

-1 - 1,155 (1,156 bases shown) - +

Primer pair 1
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer CACTGACTGACCGCCTGAAT Plus 20 84 103 60.04 55.00 3.00 2.00
Reverse primer CTCGCGGGTGTTTCGTTTTT Minus 20 165 146 59.97 50.00 4.00 0.00
Internal oligo Plus
Product length 82
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron
size
Products on intended target

2 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 82
Forward primer 1 CACTGACTGACCGCCTGAAT 20
Template 84 .................... 103

Reverse primer 1 CTCGCGGGTGTTTCGTTTTT 20

Template 165 .................... 146

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 2
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer CAACTTAGGCTGGGCCATGA Plus 20 566 585 60.03 55.00 5.00 2.00
Reverse primer GTGGCAGAAGCAGGAGAGTT Minus 20 940 921 59.96 55.00 3.00 0.00
Internal oligo Plus
Product length 375
Product Tm
Product Tm -
min(OLIGO
Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 375

Forward primer 1 CAACTTAGGCTGGGCCATGA 20
Template 566 .................... 585

Reverse primer 1 GTGGCAGAAGCAGGAGAGTT 20

Template 940 .................... 921

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 3
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer TCGCCCTCATTATCGGCATC Plus 20 469 488 60.04 55.00 3.00 0.00
Reverse primer GGGGGTGAGGGTTTGCATTA Minus 20 842 823 59.96 55.00 4.00 2.00
Internal oligo Plus
Product length 374
Product Tm
Product Tm -
min(OLIGO Tm)

3 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 374

Forward primer 1 TCGCCCTCATTATCGGCATC 20
Template 469 .................... 488

Reverse primer 1 GGGGGTGAGGGTTTGCATTA 20

Template 842 .................... 823

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 4
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer GATCGCCCTCATTATCGGCA Plus 20 467 486 60.04 55.00 4.00 0.00
Reverse primer TGTTTTGGTGGCCATTGCAG Minus 20 689 670 59.89 50.00 6.00 2.00
Internal oligo Plus
Product length 223
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 223

Forward primer 1 GATCGCCCTCATTATCGGCA 20
Template 467 .................... 486

Reverse primer 1 TGTTTTGGTGGCCATTGCAG 20

Template 689 .................... 670

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 5
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer ACAATGGTTGGAGGCTGAGG Plus 20 495 514 59.96 55.00 4.00 1.00
Reverse primer AAAAGCCTGTGAGAGGTGGG Minus 20 792 773 59.89 55.00 5.00 0.00
Internal oligo Plus
Product length 298

4 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

Product Tm
Product Tm -
min(OLIGO
Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 298

Forward primer 1 ACAATGGTTGGAGGCTGAGG 20
Template 495 .................... 514

Reverse primer 1 AAAAGCCTGTGAGAGGTGGG 20

Template 792 .................... 773

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 6
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer TCAATTGCTGGACTCCCACC Plus 20 759 778 59.96 55.00 6.00 3.00
Reverse primer TGGAGTTGGGCAACGTCATT Minus 20 971 952 60.18 50.00 6.00 3.00
Internal oligo Plus
Product length 213
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 213

Forward primer 1 TCAATTGCTGGACTCCCACC 20
Template 759 .................... 778

Reverse primer 1 TGGAGTTGGGCAACGTCATT 20

Template 971 .................... 952

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 7
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%

5 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

strand complementarity complementarity

Forward primer CAAAAACGAAACACCCGCGA Plus 20 145 164 60.25 50.00 4.00 2.00
Reverse primer GATGCCGATAATGAGGGCGA Minus 20 488 469 60.04 55.00 3.00 0.00
Internal oligo Plus
Product length 344
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 344

Forward primer 1 CAAAAACGAAACACCCGCGA 20
Template 145 .................... 164

Reverse primer 1 GATGCCGATAATGAGGGCGA 20

Template 488 .................... 469

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 8
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer GCAATGGCCACCAAAACACT Plus 20 672 691 59.89 50.00 6.00 1.00
Reverse primer AGGATGGTTGTTCCGGTTGG Minus 20 742 723 60.25 55.00 4.00 0.00
Internal oligo Plus
Product length 71
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 71
Forward primer 1 GCAATGGCCACCAAAACACT 20
Template 672 .................... 691

Reverse primer 1 AGGATGGTTGTTCCGGTTGG 20

Template 742 .................... 723

Products on allowed transcript variants

Products on potentially unintended templates

6 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

Products on target templates

Primer pair 9
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward primer AACTCTCCTGCTTCTGCCAC Plus 20 921 940 59.96 55.00 3.00 1.00
Reverse primer TGCATTCGGGTGATGTGAGT Minus 20 1151 1132 59.68 50.00 4.00 1.00
Internal oligo Plus
Product length 231
Product Tm
Product Tm -
min(OLIGO Tm)
Exon junction
Total intron size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 231

Forward primer 1 AACTCTCCTGCTTCTGCCAC 20
Template 921 .................... 940

Reverse primer 1 TGCATTCGGGTGATGTGAGT 20

Template 1151 .................... 1132

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

Primer pair 10
Template Self Self 3'
Sequence (5'->3') Length Start Stop Tm GC%
strand complementarity complementarity
Forward
CAATTGCTGGACTCCCACCT Plus 20 760 779 59.96 55.00 6.00 1.00
primer
Reverse primer CTGTGGCAGAAGCAGGAGAG Minus 20 942 923 60.39 60.00 3.00 0.00
Internal oligo Plus
Product length 183
Product Tm
Product Tm -
min(OLIGO
Tm)
Exon junction
Total intron
size
Products on intended target

>AF288233.1 Draco spilopterus TNHC 57775 tRNA-Met gene, partial sequence; NADH dehydrogenase subunit 2 (ND2) gene,
complete cds; tRNA-Trp gene, complete sequence; and tRNA-Ala gene, partial sequence; mitochondrial genes for mitochondrial
products

product length = 183

Forward primer 1 CAATTGCTGGACTCCCACCT 20
Template 760 .................... 779

Reverse primer 1 CTGTGGCAGAAGCAGGAGAG 20

Template 942 .................... 923

7 of 8 11/24/2016 7:58 PM
Primer-Blast results https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi

Products on allowed transcript variants

Products on potentially unintended templates

Products on target templates

8 of 8 11/24/2016 7:58 PM