Name : Rahma Eka Kartika

ID Number : K4320066
Class : A
Species : Annona squamosa (MK450456.1)
Gene Target : matK

1. Primer candidate
Table 1. Design Primer products based on sequence MK450456.1

No O Squence Length Tm GC Product

Start
(oC) % size
F tctcttctggcgtcaaaggg 20 413 59,7 55
As1 332
R cgggccggcttactaatagg 20 744 60 60
F accccgctcatctggaaatc 20 24 59,8 55
As2 418
R agatgagtcccctttgacgc 20 441 59,7 55
F ggcgtcaaaggggactcatc 20 421 60,7 60
As3 345
R tcagaatcggacgaatcggc 20 765 60,2 55
F ggagctgttcttgagcgaac 20 281 59,2 55
As4 317
R actgcggaagaattttggcg 20 597 59,8 50
F accttaccccgctcatctgg 20 19 61,3 60
As5 500
R tcctgttcggttgagaccac 20 518 59,6 55
F cttctggcgtcaaaggggac 20 416 61 60
As6 324
R cggcttactaatagggtgccc 21 739 60,3 57,1
F tgcgattctttctccacgac 20 96 58,3 50
As7 338
R cccctttgacgccagaagag 20 433 60,7 60
F ctttggagctgttcttgagcg 21 277 59,8 52,4
As8 319
R tgcggaagaattttggcgtac 21 595 59,8 47,6
As9 F ccgctcatctggaaatcttgg 21 27 59,1 52,4 496
R tggatcctgttcggttgagac 21 522 59,7 52,4
As10 F cgatcaacatcctttggagctg 22 266 59,6 50 742
R ggccggcttactaatagggtg 21 742 60,3 57,1
Pt-n: Name of primer candicates, O: Oligo, F: forward primer, R: reverse primer, Length:
Length of primer, GC%: Guanin-cytosine chain percentage, and Tm: melting temperature,

2. Quality test

Table 2. Quality test with Oligoanalyzer

Hetero
Hairpin Self dimer
Product Tm dimer
No O Sequence GC% (ΔG (ΔG
size (°C) (ΔG
kcal/mole) kcal/mole) kcal/mole)
TCT CTT CTG GCG
F TCA AAG GG 332 57 55 1,43 -3,61
-6,21
CGG GCC GGC TTA
As1 R CTA ATA GG 57,5 60 1,42 -16,3
ACC CCG CTC ATC
0,31
F TGG AAA TC 418 57,2 55 -3,61
-9,77
AGA TGA GTC CCC
As2 R TTT GAC GC 57,1 55 0,74 -3,61
GGC GTC AAA GGG
F GAC TCA TC 345 58,2 60 -0,61 -3,61
-6,53
TCA GAA TCG GAC
As3 R GAA TCG GC 57,2 55 0,6 -5,19
GGA GCT GTT CTT
F GAG CGA AC 317 56,2 55 -1,02 -6,34
-7,06
ACT GCG GAA GAA
As4 R TTT TGG CG 56,5 50 -0,49 -5,36
ACC TTA CCC CGC
F TCA TCT GG 500 59,1 60 1,21 -3,61
-6,68
TCC TGT TCG GTT
As5 R GAG ACC AC 56,9 55 -1,66 -4,41
CTT CTG GCG TCA
F AAG GGG AC 324 58,4 60 -0,07 -3,61
As6 -6,21
CGG CTT ACT AAT
R AGG GTG CCC 57,4 57,1 -1,15 -6,21
TGC GAT TCT TTC
F TCC ACG AC 338 55,1 50 -0,56 -3,61
As7 -6,75
CCC CTT TGA CGC
R CAG AAG AG 58,1 60 0,93 -3,61
CTT TGG AGC TGT
F TCT TGA GCG 319 56,4 52,4 0,28 -6,34
As8 -7,06
TGC GGA AGA ATT
R TTG GCG TAC 56,2 47,6 0,68 -5,36
 CCG CTC ATC TGG 496
F AAA TCT TGG 55,9 52,4 1,4 -3,61
As9 -6,68
TGG ATC CTG TTC
R GGT TGA GAC 56,7 52,4 0,74 -10,76
CGA TCA ACA TCC 742
F TTT GGA GCT G 56,1 50 0,87 -6,34
As10 -4,74
GGC CGG CTT ACT
R AAT AGG GTG 57,4 57,1 1,07 -16,03
Pt-n: Name of primer candicates, O: Oligo, F: forward primer, R: reverse primer, , GC%:
Guanin-cytosine chain percentage, and Tm: melting temperature
Analisis primer terbaik:
Dasar pengambilan nilai terbaik yaitu dilihat dari nilai TM berkisar 52-58oC, nilai GC
berkisar 40%-60%, hairpin lebih dari -3, serta nilai self dimer melebihi -6.
3. Specifity test (in silico)

Forward primer menempel pada template 1764 hingga 1745, sedangkan reverse
primer menempel di template 1347 hingga 1366.
4. PCR in silico

ACCCCGCTCATCTGGAAATCttggttcaaatacttcactcttggatacaagatgctccctctttacatttattgcgattc
tttctccacgactatcagaattcgaatagtctcaaagctcaaaagaaatccatttccgtttgttcaaacgagaatcgaag
attcttcttgttcatatataatattcatgtatatgaatgtgaatcggtatttatttttctccgtaaacaatcttttcatt
tacgatcaacatcctttggagctgttcttgagcgaacacatttctatggaaaaatagaacatcttgtagtagtgcttcgg
aatgattttcagaagaccttatggatgttcaaagacccttttatgcattatgtcagatatcaaggaaattctcttctgGC
GTCAAAGGGGACTCATCT

Panjang Fragment:418 nt