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primer analysis.pdf
- 1. Sequence of RiceGADPH GGCTGCTGCTGCATTCTTCTTGATCTGATCGTCGGACATGGCGTCGCCCATGCTCTCCGCCGCCACCGTG CCACTCCAGGGAGGCGGCTTGTCGGAGTTCTCCGGCCTGAGGAGCTCGTCGTCGCTGCCGCTGCGGCGGA ATGCCACCTCCGACGACTTCATGAACGCGGTCTCCTTCAGGACCCACGCGGTTGGCACGAGCGGCGGGGC GCGGCGGGCGCCGACGGAGGCGAAGCTGAAGGTGGCGATCAACGGGTTCGGCCGCATCGGGCGCAACTTC CTGCGGTGCTGGCACGGGCGCGGCGACAGCTCGCCCCTCGACGTCATCGCCATCAACGACACCGGAGGCG TGAAGCAGGCGTCGCACCTCCTCAAGTACGACTCCACGCTCGGCATCTTCGACGCCGACGTCAAGCCCGT GGGCGACAACGCCATCTCCGTCGACGGCAAGGTGATCAAGGTCGTCTCCGACCGCAACCCGTCCAACCTG CCGTGGGGCGAGCTCGGCATCGACCTCGTCATCGAGGGCACCGGTGTGTTCGTCGACCGCGACGGCGCGG GCAAGCACATCCAGGCCGGCGCCAAGAAGGTGCTCATCACCGCCCCCGGCAAGGGTGACATCCCCACCTA CGTCGTCGGCGTCAATGCCGACCAGTACAGCCCCGACGAGCCCATCATCAGCAACGCCTCCTGCACCACC AACTGCCTCGCCCCCTTCGTGAAGATCCTCGACCAGAAGTTCGGCATCATCAAGGGGACGATGACCACCA CCCACTCCTACACCGGCGACCAGAGGCTGCTCGACGCGAGCCACCGTGACCTCCGGCGCGCGCGTGCGGC GGCGCTGAACATCGTGCCGACGTCCACCGGCGCGGCCAAGGCCGTGGCGCTGGTGCTCCCGAACCTCAAG GGGAAGCTCAACGGGATCGCGCTCCGGGTGCCGACGCCGAACGTGTCCGTGGTCGACCTCGTGGTGCAGG TGTCCAAGAAGACCCTCGCCGAGGAGGTGAACCAGGCGTTCCGCGACAGCGCCGCCAACGAGCTCAAGGG CATCCTCGAGGTCTGCGACGTGCCGCTCGTGTCCGTCGACTTCAGGTGCTCCGACGTGTCGTGCACCATC GACGCGTCGCTCAGCATGGTCATGGGCGACGACATGGTGAAGGTCATCGCCTGGTACGACAACGAGTGGG GATACTCGCAGAGGGTCGTCGACCTCGCCGACATCGTCGCCAACCAGTGGAAGTGAGAACGTAGAAGCTG CTGCTGCTGCTGCCTTTGTAACTCCACCTCGCTCCAAAAATTTTCCCCAGACTTCTTCCTTCTTCCTCTC GATCCTTTGTAATGAGAATTTCGAAGTTTCGAGCGCCCAACACAGTTTCTTGTTTCTTTCTTTGCTTTTC TTCCCCACCTCATCTGACTTTTCACTGTCCGGCACGGATTATTATCGACTTCCGGCAATTGGAAATCGCA ATGCTGTGTAATGTTCATGTGTAATACCGTATATACTACCGGACACCGGTTGCCAGGTTCTGGTATAGAA TTTCAGCATGTTGCTTGGGACCATTTTTGCT
- 2. Internal Primers usingprimer3 No mispriming library specified No hyb oligo mishyb library specified Using 1-based sequence positions OLIGO start len tm gc% any 3' seq LEFT PRIMER 353 25 58.44 60.00 6.00 1.00 AAGCAGGCGTCGCACCTCCTCAAGT RIGHT PRIMER 540 25 58.13 60.00 8.00 3.00 AACACACCGGTGCCCTCGATGACGA HYB OLIGO 426 25 58.64 60.00 10.00 8.00 ACAACGCCATCTCCGTCGACGGCAA SEQUENCE SIZE: 1571 INCLUDED REGION SIZE: 1571 PRODUCT SIZE: 188, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 1 GGCTGCTGCTGCATTCTTCTTGATCTGATCGTCGGACATGGCGTCGCCCATGCTCTCCGC 61 CGCCACCGTGCCACTCCAGGGAGGCGGCTTGTCGGAGTTCTCCGGCCTGAGGAGCTCGTC 121 GTCGCTGCCGCTGCGGCGGAATGCCACCTCCGACGACTTCATGAACGCGGTCTCCTTCAG 181 GACCCACGCGGTTGGCACGAGCGGCGGGGCGCGGCGGGCGCCGACGGAGGCGAAGCTGAA 241 GGTGGCGATCAACGGGTTCGGCCGCATCGGGCGCAACTTCCTGCGGTGCTGGCACGGGCG 301 CGGCGACAGCTCGCCCCTCGACGTCATCGCCATCAACGACACCGGAGGCGTGAAGCAGGC >>>>>>>> 361 GTCGCACCTCCTCAAGTACGACTCCACGCTCGGCATCTTCGACGCCGACGTCAAGCCCGT >>>>>>>>>>>>>>>>> 421 GGGCGACAACGCCATCTCCGTCGACGGCAAGGTGATCAAGGTCGTCTCCGACCGCAACCC ^^^^^^^^^^^^^^^^^^^^^^^^^ 481 GTCCAACCTGCCGTGGGGCGAGCTCGGCATCGACCTCGTCATCGAGGGCACCGGTGTGTT <<<<<<<<<<<<<<<<<<<<<<<<< 541 CGTCGACCGCGACGGCGCGGGCAAGCACATCCAGGCCGGCGCCAAGAAGGTGCTCATCAC 601 CGCCCCCGGCAAGGGTGACATCCCCACCTACGTCGTCGGCGTCAATGCCGACCAGTACAG 661 CCCCGACGAGCCCATCATCAGCAACGCCTCCTGCACCACCAACTGCCTCGCCCCCTTCGT 721 GAAGATCCTCGACCAGAAGTTCGGCATCATCAAGGGGACGATGACCACCACCCACTCCTA
- 3. 781 CACCGGCGACCAGAGGCTGCTCGACGCGAGCCACCGTGACCTCCGGCGCGCGCGTGCGGC 841 GGCGCTGAACATCGTGCCGACGTCCACCGGCGCGGCCAAGGCCGTGGCGCTGGTGCTCCC 901GAACCTCAAGGGGAAGCTCAACGGGATCGCGCTCCGGGTGCCGACGCCGAACGTGTCCGT 961 GGTCGACCTCGTGGTGCAGGTGTCCAAGAAGACCCTCGCCGAGGAGGTGAACCAGGCGTT 1021 CCGCGACAGCGCCGCCAACGAGCTCAAGGGCATCCTCGAGGTCTGCGACGTGCCGCTCGT 1081 GTCCGTCGACTTCAGGTGCTCCGACGTGTCGTGCACCATCGACGCGTCGCTCAGCATGGT 1141 CATGGGCGACGACATGGTGAAGGTCATCGCCTGGTACGACAACGAGTGGGGATACTCGCA 1201 GAGGGTCGTCGACCTCGCCGACATCGTCGCCAACCAGTGGAAGTGAGAACGTAGAAGCTG 1261 CTGCTGCTGCTGCCTTTGTAACTCCACCTCGCTCCAAAAATTTTCCCCAGACTTCTTCCT 1321 TCTTCCTCTCGATCCTTTGTAATGAGAATTTCGAAGTTTCGAGCGCCCAACACAGTTTCT 1381 TGTTTCTTTCTTTGCTTTTCTTCCCCACCTCATCTGACTTTTCACTGTCCGGCACGGATT 1441 ATTATCGACTTCCGGCAATTGGAAATCGCAATGCTGTGTAATGTTCATGTGTAATACCGT 1501 ATATACTACCGGACACCGGTTGCCAGGTTCTGGTATAGAATTTCAGCATGTTGCTTGGGA 1561 CCATTTTTGCT KEYS (in order of precedence): >>>>>> left primer <<<<<< right primer ^^^^^^ hyb oligo ADDITIONAL OLIGOS start len tm gc% any 3' seq 1 LEFT PRIMER 353 25 58.44 60.00 6.00 1.00 AAGCAGGCGTCGCACCTCCTCAAGT RIGHT PRIMER 543 25 58.13 60.00 8.00 2.00 ACGAACACACCGGTGCCCTCGATGA HYB OLIGO 426 25 58.64 60.00 10.00 8.00 ACAACGCCATCTCCGTCGACGGCAA PRODUCT SIZE: 191, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 3.00
- 4. 2 LEFT PRIMER354 25 57.76 60.00 6.00 2.00 AGCAGGCGTCGCACCTCCTCAAGTA RIGHT PRIMER 543 25 58.13 60.00 8.00 2.00 ACGAACACACCGGTGCCCTCGATGA HYB OLIGO 426 25 58.64 60.00 10.00 8.00 ACAACGCCATCTCCGTCGACGGCAA PRODUCT SIZE: 190, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 3 LEFT PRIMER 354 25 57.76 60.00 6.00 2.00 AGCAGGCGTCGCACCTCCTCAAGTA RIGHT PRIMER 540 25 58.13 60.00 8.00 3.00 AACACACCGGTGCCCTCGATGACGA HYB OLIGO 426 25 58.64 60.00 10.00 8.00 ACAACGCCATCTCCGTCGACGGCAA PRODUCT SIZE: 187, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 4 LEFT PRIMER 353 26 57.89 57.69 6.00 2.00 AAGCAGGCGTCGCACCTCCTCAAGTA RIGHT PRIMER 543 25 58.13 60.00 8.00 2.00 ACGAACACACCGGTGCCCTCGATGA HYB OLIGO 426 25 58.64 60.00 10.00 8.00 ACAACGCCATCTCCGTCGACGGCAA PRODUCT SIZE: 191, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00
- 5. External primers usingOligonucleotide calculator Reverse Primer Forward Primer
- 6. Hairpin Loop ofForward primer from idtprimeranalysis
- 7. Self-dimer of Forwardprimer
- 8. Hairpin loop ofreverse primer idtprimeranalysis Self-dimer of Reverse primer
- 9. Restriction Analysis frommolbiotools Restriction Sites Restriction enzymes
- 10. Restriction fragments using3 unique restriction enzymes Bands of ApaLI , XhoI and NaeI
- 11. Circular structure usingNEBcutter Single cutters
- 12.