Primer Designing and Validation

Objective: The purpose of this lab is to help you to design primers to extract Elicitin genes. We will use ClustalW to align the homologs nucleotide sequences to nd well-conserved regions. The next step will be to extract a conserved region from the aligned nucleotide sequences and design primers. Next step is to validate the primers using OligoCalc and FASTPCR. Detecting Conserved regions using ClustalW 1. Access ClustalW homepage at http://www.ebi.ac.uk/Tools/clustalw2/index.html 2. Paste the sequences given below in text box and Click the Run button. 3. In the result page click start JalView Button 4. Alignment will open in an Multiple alignment editor 5. In Multiple alignment editor choose Color: percent Identity 6. Note the first conserved region. 7. Open OligoCalc at http://www.basic.northwestern.edu/biotools/oligocalc.html. 8. Enter the conserved nucleotide sequence and click Calculate button 9. Note down the primer properties, if it satisfies the GC% and Tm range. 10. Click the check self-complementarity button. 11. Check if there is any hairpin and primer dimmer formation. 12. Choose this conserved region as forward primer. 13. Choose another conserved region for reverse primer from alignment editor, such that it will have a reasonable amplicon size in between. 14. Find the reverse complement of this conserved region to get the 3 to 5 reverse primer. 15. Open molecular tool kit from http://www.vivo.colostate.edu/molkit/index.html 16. Choose Manipulate and Display Sequences. Paste the conserved region and click inverse complement button. 17. Note the reverse complement sequence and check with OligoCalc. 18. Choose this conserved region as Reverse primer.
>seq1 AAGTACTGTACCAGTGGTGGACCATTTCTAAATACAAATCATGGACGTGGACGCTCTCTTGTGAAGGCGT CATTCCTCTGTTGCAGGACCAGATACGCGTCTGTCTCTCGGTCTGCCGCAGGATTCATGCCACGTGAACG

TTTTCGGTGCTTCGACAACTTTTTACAGTCGCGACGATGTCGCCGACGAAATCCAAAGAGGCAAAACCTG TCAATGTCGTTCCAAGGCAATGCGAGTCAAGATGACATTGTTTGCTGACCCTTCCAGTCCTAGCTCCACA ACTTCCATTGTGCAATTTGCTCTGTTATCCCCACCCACTCCTCCTCACTCCGTCCACGATGAACTTTCGT GCTCTGTTCGCTGCCACCGTTGCTGCCCTCGTCGGCTCCACTTCCGCCACCACGTGCACCACCTCGCAGC AGACCGTAGCGTACGTGGCGCTCGTAAGCATCCTCTCGGACACGTCGTTTAATCAGTGCTCGACGGACTC CGGCTACTCGATGCTGACGGCCACCTCGCTGCCCACGACGGAGCAGTACAAGCTCATGTGCGCGTCGACG GCGTGCAAGACGATGATCAACAAGATCGTGTCGCTCAACGCTCCCGACTGCGAGCTGACGGTGCCAACTA GTGGCCTGGTACTCAACGTGTACTCGTACGCGAACGGGTTCTCGTCTACGTGTGCGTCGCTATGAGTGGA CTTGACTCAGCGCGCTCAGCCGCTGAGTCAGCGTCCATTGGCCGTATCATCTTGTCTTTCATCGACGATT AGGCTTTGTTCTCCGCGTCTGTTGCTATTACTGTAATAGTGTAGCAGCTGCGGTATAAAGCTATTTGAAT AAAAATTCTTGTATGCCATACTTGTTGCTGTAGCTCGGGTGCGTTGTTTGTTCAATGCAG >seq2 GTCGACGAAAGCCGAAGTGCGTGGCAGATCTTGCCGTTCGAATGCTACGCGCCACGGCAAAACCTACACG GTACAACAGCTTCAAATAAACCTGCAAGCGAGCCGCCAGCCCAACTCCAGCTAGTCAAGCCTAGTTTGCC TCCAACTGCCATTGTGCAATTTGCTCTCATCCACACCCACCCCACTTCTCCCCCACCTCATCCGAAATGA ACTTCCGCGCTCTGTTCGCCGCCACCGTCGCCGCCCTCGTCGGCTCCACCTCCGCCACCACGTGCACCAC CACGCAGCAAACTGCGGCGTACGTGGCGCTCGTAAGCATCCTCTCGGACACGTCGTTCAACCAGTGCTCG ACGGACTCTGGCTACTCAATGCTGACGGCCACCTCGTTGCCCACGACGGAGCAGTACAAGCTCATGTGCG CGTCGACGGCGTGCAAGACGATGATCAACAAGATCGTGACGCTGAACCCGCCCGACTGCGAGTTGACGGT GCCTACGAGCGGCCTGGTACTCAACGTGTTCACGTACGCGAACGGGTTCTCGTCTACGTGCGCGTCACTG TAAGCGGGTTTGATCTCTGCGTCCAGAATCGAT >seq3 ATGAACTTCCGCGCTCTGTTCGCCGCCACCGTCGCCGCCCTCGTCGGCTCCACCTCCGCCACCACGTGCA CCACCACGCAGCAAACTGCGGCGTACATGGCGCTCGTAAGCATCCTCTCGGACACGTCGTTCAACCAGTG CTCGACGGACTCTGGCTACTCAATGCTGACGGCCACCTCGTTGCCCACGACGGAGCAGTACAAGCTCATG TGCGCGTCGACGGCGTGCAGGACGATGATCAACAAGATCATGACGCTGAACCCGCCCGACTGCGAGTTGA CGGTGCCTACGGGCGGCCTGGTACTCAACGTGTTCACGTACGCGAACGGGTTCTCGTCTACGTGCGCGTC ACTGTAA >seq4 CGCGATCGACGGTCAACTTCAGGCTTCACCTGTGGCATGGCACCTTSGATCGAACAGTGACTGTCAGGTG TAGACGTCAGAGAACTGAGCTGTTCGACAGCGTCTTCAGACCGCCTTTCCACTTGGTCCGAGTCAAGGTC GCGTCGAGATCAGCCGAGCGCCCGCCACACGGGCCTACGGCTAAGGAAGGCCCTCTGCTAGGATAGCAGC TTTTGCCAGATAGTGCGATTGCGTCTTGGGCTGTTCTGGTTCCTCTTGCTCATTCTGCAATTGGCTCCGT CACACGCACGACATCCACTCGCCACCTCCACCCAAGATGAACTTCACCGCTCTGCTCGCTGCCGTCGCCG CCGCCCTCGTCGGCTCCGCCAACGCCACGGCCTGCACGGCCACCCAGCAGACCGCCGCGTACAAGACGCT CGTGAGTATCCTGTCCGAGTCGTCCTTCTCGCAGTGCTCCAAGGACTCGGGCTACTCCATGCTGACGGCC ACGGCACTGCCCACCAACGCGCAGTACAAGCTCATGTGCGCGTCCACGGCCTGCAACACCATGATCAAGA AGATCGTCGCGCTGAACCCTCCTGACTGCGACCTGACCGTCCCGACCAGCGGTCTCGTGCTGGACGTGTA CACGTACGCCAACGGCTTCTCGTCCAAGTGCGCGTCGCTCTAAGTGTGTCCTCAAACTTGATGGCGCTCC GAGTTTTGAACGCGAGGCTCCCCATTGCGTACCTTCTCTCTCTGTATCGGCTAGCCATTTACTGAGGATG GAGGAGCAGTCAATATTTCGCACAAACTTTCGAAGTACTATGTGAATGAACTGGTATGAATTTTGTTTAT CTACGCGTTCTCTTTTGTGAGTTGGCGTGCTTGAGTGTGAGCGCACCTGCAATCACAGTTTTGACGCGGA CCTGGCTTACCTTCGTAACAACAGAAAGCTGCCCACACTCCAGCCACGCCATCCATTTTCATTTTCGGTT ACAGTGAATTAGTAGTTATTTGTGCTAACTACAGCCAGAGCATCAACAGGCGGCGCACTATGGGGATTCC GGTGTTGTGCTCCTTCCATGGTTGCATCTCCGCAAGATCTTGCCGTTTCTCATTGCGTCACTTGTTGATC CATCGAACAGCAAGAGCGCTAAGCATTTCAATGACATCTGCGTGGAATCAGCGGGGCCGTAACGATATCA TGCCGCATCTTCGGCAAGCCGTCTAGCCATTGAGGTGCTGAACGACTGTCTTCTTGTGCTATCCGGTCTT CAACGAGACTGCGCAACTCCAGGATCACCTGAGAGCAACAGGGAACGGACTCGCTGAGCCTGATGAAGGA GGGGGATATGCTCGGCATGCGTCCGCTGCGACAGCTTGTCCACAGCTCCAGTCATTGCGTGCTATTGTAT ATGACGTCTTTGTGAGCTACCGATCCGCTCACGGATACATCCTTGCCTGCGCCTACGATGCCTGCTAGAA GTAACGGGTCCTTGTAACCTGGCCGCTGCTGGTGGCTGTTCCCCAGGCAGCAGAGCCCAACGACCGCCTC CTGCAGGTGCATCACAACATAGCCGTCCATTGCCTCCGCACTCCACAATTTGAAATTTGCCTTGCCAACC ACAAGCCTCCACCACACCCACCACTCAAGCCCCACTTCCGCTCCCCAAGATGAACTTCGCCACCCTCCTC GCTGCCACCGCCGCCGCCCTCGTCGGATCCGTCTCCGCCACGGCCTGCACTTCCACCCAGCAGACCGCCG

CCTACGTCGCGCTCGTGAGCATCCTCTCGGAGTCGTACTTCTCAACGTGCGCCAGCGACTCGGGCTACTC CATGCTGACGGCCACGGCGCTTCCCACGACGGCTCAGTACAAGCTCATGTGCGCGTCGACCGCGTGCCAG GAGATGATCACGGAGATCATCGCGCTGGACCCGCCCGACTGCGACCTGACGGTGCCGACGAGCGGTTTGG TGATCAACGTGTACGAGTACTCGAACGACTTTGTGTCGACGTGCGCGTCGCTGTCGTCCTCGGCGTCGTC TTAAGCGCTGAAGCGCTGGTGGCGGTCGCTTCACCGGCCGTGACGTCACTGGACTGAACGAATGTTCAAG GGTTGTCGCTTTTGGTAGCGCCACGATGTCCAGACTGAAGTTTCAGGCGCTTCTTACGGGAATGGCCCGC CCGCTTGGTTATGAGGGCGAGCTTTATGAATGCACAGTAACGATCGAATGAATTGACGTTTGACTCTGTT AACCCGTTGTTGGTGCATTTGATACGAGCCTGCGTGTTGGTCTTCACCTCTGCCTTCGTCATCACATCAC ATTTGCTTGGTAAATCACGTCAAAAGGGTGAAGCGTGAGCTTTCAATGAAAACCTTACATTCTGCAATCG CATCAGTCTCGATCACCGAAAAGATACGAATCTGTTCATATTGAAGTGACACAGCCAATCAGATCGAGTM TACGCTACAGTGCGAGTTGCATGTTTTTATCGGTGGACGTCAAATGAGGCGGGTCTGCAGATGTTTGATG TTGCTAGACCAACTTGTTCGCGATTCGGCCTGTTGCTCTTACATGATACCACAATATACCGGTTCCGCTG CTGTTCTCGTAGCTACAAGATCTATCTGTCGCGTAAGAGAGCACTGAACGGAGAATCGACAGTAAAGCAC GATGAAGTGGTGGGCCATGAATCGCTGTGCACATCATCCGCCCTTCGGTCCACCACACACTGTGACTGGA AGGCACTAGCGAACGNTAGGTTGTCGCCCCCGCGCTGAAACCAGAAGAACGGGATTGTATTGTGACCAGC TGCCACCACAAACTCAACGCACGTCGAGACAAACGTTTTCGGTCCCCGTTCAAGTGGAACTTCCACCCAC AGGCTGTGCATCAACTGCGTCCACCCGATTAGCTTATTTATCTCTTTCTGCTTCAAGCAGGTTGCTTACC AAGTGGTCGAGAGCGGCTGGCATCAGGTGAGCCAAACTATTGTCGGCTGCGACTTGCTTGGCAGTGCCAA GTTGACAGACGCGTAACGCGGGCTGACTGGGTTCGCTGCATCCATCTTTTCACTCGCAGTCGATGTGACC TCACTGGGGCCTCCCGAGGCAGTGCGAGAGCTGGCGACCAGGAGTGGATCTCGACCCGTTAGGCAACGGA TCCCTTCAAGCAGCATCCATGCATCGTGGCGAGACAGCGTGGTGTGCGTGTGTGTGTGGTTATCAGGTGT TGATCCAAAGTGCAGGACCCCCCATCTTGTCTTCGGCAGCCGAAGTTCGGCTTCATGTCCGTAGAGTGGC ATGTATCCTGTCAGTCGGCGGCACTGCTGCCAACAGATCTTCAAACAAAGCGGGGCAAGCGCGCGGTGCA GCCAAGCGGTTCACTGCGTGAGTCCATAGGCTACAAATGCTGCGCTGCGGCTGCGGAAGACGCGAGAAAC TGCTCTGGGACAGCCGTTCGTCCGGGCAGCGCGTGACATCCTCGTCAACGGCCACGGCCCTCGTTCCCAG GACGCTGATGCCTGCCTATCTTGCGATACACTGCTCTAAAGTACGTACGCAGTGCATGTTGCATGCAACC TTAGCATACTGCTGTATCGAGGCTGCACTGCACCCAACGCACGGTTCGCTGAATTTGCAGTGTCGGTCCC TGAAACTTTCAGGCTGAAAGACATCCTGGCGATTTCATGAGCACGAAAGATGTCTGCCGGCCGGCAAGGT GGACTCTTTCACAGCGGATCCCGCGAGACGCTTCTCTCGCCCAGGACCAGATGCGCGCCCCTCTCTCGTC CTACGGCGGATCCAGCCAAAATTGATCACGCTCGATCACGCTGGCAGCATGACACAGCAGCATGATGCTG CTCGACGGCCCGCTCTGCTCGTCTCGGCAGTCGCCGACAGCGGAAAGCTTTGCCGTTCGAACTGCGCGCG CAAGCCACGGCAACGCCTTACCCAGAGCCATCGGCGTTAATTCACCCTCGCACCAACGCCGCCGAGTCAA GATAACATGACACGACCTGCCACCGCCACCGCTGCTGGCATTGTGCAATTTGCTCTGTCGCCCACACCCC ACCAGCACCCTTCCTCCCCACCACCTCCACGATGAACTTCCGCGCTTTGTTCGCCGCCACCGTCGCCGCG CTCGTCGGCTCCACCTCGGCCACCACGTGCACGTCGACGCAGCAGACCGCCGCGTACGTGGCTCTGGTGA GCATCCTCTCGGACTCGTCGTTCAGCCAGTGCGCGACGGACTCGGGCTACTCGATGCTGACGGCCACCTC GCTGCCCACGACGGCGCAGTACAAGCTCATGTGCGCGTCGACGGCGTGCAACACCATGATCAAGAAGATC GTGACGCTGAACCCTCCTGACTGCGAGCTGACGGTGCCCACCAGCGGCCTGGTGCTCAACGTGTACTCGT ACGCGAACGGCTTCTCGGCCACGTGCGCGTCGCTGTAAGCAGCTTCGTTCGTGTGAGTCGCCACACTGCG TCCAGCGTTTCATCTATAAGGTGCCGATGCGTGTGGCATGCTGACTTGTCTTTGGGTCAAGTACGCAGCT TGAGCAGATAGTTTCGGCGTAAGTTGGCTGTCGCTCGAGCGTTTGTAAAGCTTTTGAATGAAGATTTTTG TCATACTTTCTTCACGTGCTATCGGTTTTTCATCTTAAACTCCACTACMTGTATCATTTTTTTTTTCAAT ATCGATYCACTTGTTCGCTGCCACGGGTACGTACAGTAAGTATATGACTACGGRCTCSAAACCTCGAGAC GTGATACAGCTCTGTGCACGAACAGCAATAGCACCAYGTTGGATGTTGCTCGTCCTGACARGGTTTGCAT CRCTGCTTCTCGTCCTATCCTGCCTCCGTGGTGACTGCTYCTGACACYGTGGGGCAAGAGCAGAATCGGA TATSCTCAGCATGGCGATCGATCCAATGCGTCTGGTTCGAAATCAGCATCGATCAAGAAGCTCCTCGGTG CGACCAGAGATACTCCATGGACAGAGAATCTATAGACCCATGGCACATATCGTCACCGCTTCTCCATCTT CAGTCCTCTTCATTCTGCAATTTGCTTTGCCGATCACAAGTTTACACTGCACAAACCTCTTCACCCACCC TCCAAGATGAAGTTCACTGCTGTCCTCGCCGCCGCCGCGGCCCTCGTCGGATCCGTCAACGCCACGGCCT GCACTTCCACCCAGCAGACCGCCGCCTACGTCGCGCTCGTGAGCATCCTCTCGGAGTCGTACTTCTCAAC GTGCGCCAGCGACTCGGGCTACTCCATGCTGACGGCCACGGCACTTCCCACGACGGCCCAGTACAAGCTC ATGTGCGCGTCGACCGCGTGCCAGGAGATGATCACTGAGATCGTCTCGTTGAGCCCGCCTGACTGCGACC TGACGGTGCCGACGAGTGGCCTCGTGCTGGACGTGTACACGTACGCGAACGGCTTTACGTTGACGTGCGC GTCGCTGTCGTCGTCGTCGGCCTAAGCTTTAATGGGTGTGCCCCCTGTGGCTAGCGAAGTGCGCTGTGCT GCTATTCTCGAGTTTCTCGAGTTCAAGGGGATCCTTACTCAGACCGAAGCTACAAATGCTTCATTGCAAA AGACTTACCCGGTAAGGCGAGCTTATATGAACGCTAATGACTACGTTTCATTTTTAAATATCTCTGATGT GTTATTTTTTTTTGCTCGGCATGCGTTGCTAAG

>seq5 ATGAACTTCCGCGCTCTGCTCGCCGCCACCGTCGCCGCGCTCGTCGGCTCCACCTCGGCCACCACGTGCA CCACGACGCAGCAGACCGCCGCGTACGTGGCTCTGGTGAGCATCCTCTCGGACTCGTCGTTCAACCAGTG CGCGACGGACTCGGGCTACTCGATGCTCACGGCCACCTCGCTGCCCACGACGGCGCAGTACAAGCTCATG TGCGCGTCGACGGCGTGCAACACCATGATCACCAAGATCGTGTCGCTGAACCCACCCGACTGCGAGCTGA CGGTGCCGACCAGCGGCCTGGTGCTCAACGTGTACTCGTACGCGCACGGCTTCTCGACCACGTGCGCGTC GCTGTAAGCGACTTGGTTCGTGGCAATCCCCACGTTCAGCGCATCCACCCCCAAGACGTGGACTCGTCTT CAGATCAAGTACGCATCTTGAGCATTGTTTCAGCATTTATAAGGCTTTTGAATGAAATAAGTTTTTGTTT CCGAAAAAAAAAAAAAAA >seq6 ATGAACTTCCGCGCTCTGTTCGCCGCCACCGTCGCCGCGCTCGTCGGCTCCACCTCGGCCACCACGTGCA CCACGACGCAGCAGACCGCCGCGTACGTGGCTCTGGTGAGCATCCTCTCGGACTCGTCGTTCAACCAGTG CGCGACGGACTCGGGCTACTCGATGCTCACGGCCACGTCGCTGCCCACGACGGCGCAGTACAAGCTCATG TGCGCGTCGACGGCGTGCAACACCATGATCACCAAGATCGTGTCGCTGAACCCGCCCGACTGCGAGCTGA CGGTGCCGACCAGCGGCCTGGTGCTCAACGTGTACTCGTACGCGCACGGCTTCTCGACCACGTGCGCGTC GCTGTAAGCAGCTTGGTTCGTGGCAATCCCCCATCCACCCTCAAGACGCAGACTCACCTCTTATGCCGAC TTGTCTTCAGATCAAGTACGCAGATTGAGCAGGTAGTGTTGGCGTCGGGCGTGCATTTAACTGTTGTTTG AACATTTATAAGGCTTTTGAATGAGCAAGTTTTTGTTTCCGAAAAAAAAAAAAAAA >seq7 ATGAACTTCCGCGCTCTGTTCGCCGCTACCGTCGCTGCCCTCGTCGGCTCCACCTCCGCCGCCACGTGCA CGACCACCCAGCAGACCGCCGCGTACGTCGCCCTGGTGAGCATCCTCTCGGACTCGTCGTTCAACCAGTG CGCCACGGACTCTGGCTACTCGATGCTGACGGCCACGGCGCTGCCCACGACGGCCCAGTACAAGCTCATG TGCGCGTCGACGGCGTGCAACACGATGATCACCAAGATCGTGTCGCTGAACCCCCCTGACTGCGAGCTGA CGGTGCCCACAAGCGGTCTGGTGCTCAACGTTTACTCGTACGCGAACGGTTTCTCGTCTACGTGCGCGTC ACTGTAA

Exercise II Identify primers for the following gene sequence using Primer3 and Primer3 plus and check the properties using OligoCalc. 1. Access Primer3 at http://frodo.wi.mit.edu/primer3/ 2. Paste the sequence below 3. Set the product size range between 1500-1700. 4. Click pick primer button. 5. Check for primer properties using OligoCalc.
GACAAGAACCTGGAGATCAAGGTGACCCCGGACAAGGCCAACGGCACGCTGACCATCCAGGACTCTGGTA TTGGTATGACGAAGGCTGACCTGATTAACAACCTGGGTACGATCGCCAAGTCGGGCACCAAGGCCTTCAT GGAGGCTCTGGCTGCTGGCGCCGACATCAGTATGATTGGTCAGTTCGGTGTGGGTTTCTACTCGGCCTAC CTGGTCGCTGACAAGGTCGTGGTGCACTCGAAGCACAACGATGACGAGCAGTACGTGTGGGAGTCTGCTG CCGGTGGCTCGTTCACTGTCACGCCTGACACGTCGGAGCCCATCCAGCGCGGTACGCGCATTGTGCTGAA GCTCAAGGAGGACATGCTCGAGTACCTTGAGGAGCGCAAGCTTAAGGACCTGGTGAAGAAGCACTCGGAG TTCATTGGCTTCCCCATYAAGCTGTACGTCGAGAAGACGGAGGAGAAGGAAGTGACGGACGACGAGGAAG AGGAGGACGAGAAGGAGGGCGAGGACGACAAGCCCAAGGTCGAGGAGGTCGAAGAGGAGGAAGGCGAGAA GAAGAAGAAGACTAAGAAGATTAAGGAAGTGACCCACGAGTGGGACCACCTCAACAGCCAGAAGCCCATC TGGATGCGCAAGCCTGAAGACGTGACCCACGAGGAGTACGCGTCGTTCTACAAGTCGCTGACGAACGACT GGGAGGAGCACGCCGGCGTCAAGCACTTTTCGGTGGAGGGCCAGCTGGAGTTCAAGGCYTGTCTGTTCAC CCCCAAGCGCGCGCCGTTCGACATGTTYGAGGGCGGCGCCAAGAAGAAAGTGAACAACATCAAGCTGTAC

GTGCGTCGCGTCTTCATCATGGACAACTGCGAGGAGCTCATGCCCGAGTACCTGTCGTTCGTCAAGGGTG TCGTCGACTCGGAGGACTTGCCGCTTAACATCTCGCGTGAGACGCTGCAGCAGAACAAGATCCTGCGTGT GATCAAGAAGAACCTGGTCAAGAAGTGCCTCGAGATGTTCGCCGAACTGGCTGAGGAYAACGAGAAGTAC AACAAGTTCTACGAGTCGTTCAGCAAGAACCTCAAGCTGGGCATCCACGAGGACTCGACCAACCGCACCA AGATCGCCAAGCTGCTGCGTTACCACTCCACCAAGTCTGGCGAGGAAGTGACGTCGCTGGATGACTACAT CTCGCGTATGCCTGAGAGCCAGCCCGGTATCTACTACGTGACTGGCGAGAGCAAGAAGTCGGTGGAGAAC TCGCCATTCATTGAGAAGCTCAAGAAGAAGGGCTACGAGGTGCTGTTCATGGTGGAGGCCATTGACGAGT ACGCTGTGCAGCAGCTCAAGGAGTACGAGGGCAAGAAGCTCATCTGCGCCACCAAGGAGGGCCTCAAGAT GGAGGAGACGGAGGACGAGAAGAAGTCGTTCGAGGAGGCCAAGGCTGCCACTGAAGGACTGTGCAAGCTC ATGAAGGAGGTGCTGGACGACAAGGTGGAGAAGGTGGAGATCTCGAACCGTATTGTGGAGTCGCCCTGTG TGCTTGTTACCGGCGAGTACGGCTGGTCGGCCAACATGGAGCGCATYATGAAGGCGCAGGCTCTGCGTGA CAGCAGCACGTCGGCCTACATGTCGTCCAAGAAGACGATGGAGATCAACCCGCTGCACCCTATTATCAAG TCGCTGCGCGAGAAGGCGGAGGCTGACAAGAGCGACAAGACGGTCAAGGACCTCATCTGGCTGCTG

Exercise III Identify SSR for the sequence below using MISA and make out the flanking primers using Primer3 MISA - MIcroSAtellite identification tool. This tool allows the identification and localization of perfect microsatellite. Copy the sequence below and save in .fasta format in MISA folder. In terminal window change the directory to MISA by typing the following command: cd MISA In MISA folder open misa.ini and set the parameters as below and save the file
definition(unit_size,min_repeats): 1-10 2-10 3-6 4-6 5-6 6-6 interruptions(max_difference_between_2_SSRs): 100

Run the program by typing the following command: ./misa.pl <filename.fasta> Output: Results of the microsatellite search are stored in two files: 1. In "<FASTfile>.misa" the localization and type of identified microsatellite(s) are stored in a tablewise manner. 2. The file "<FASTfile>.statistics" summarizes different statistics as the frequency of a specific microsatellite type according to the unit size or individual motifs.

To design flanking Primers 3. Access Primer3 at http://frodo.wi.mit.edu/primer3/ 4. Paste the sequence below 5. Set the product size range between 250-300 6. Set the target position (E.g. 704, 24 requires primers to surround the 24 bases at positions 704) 7. Click pick primer button. 8. Check for primer properties using OligoCalc.
ACCACCTACTACTCAAAGCAAGCAACGAACTCAAGGCTACTTCAGCCCACCCAAACCAAA GACTCTCACCAAGACTCTCTACCGACCATGTTCAGCTCGTGGGATCTCGTCCGACCGCGC TTCTGGTACCCCATGTCGTCACTGGAACAGTCCATGATGGAGCTCGAGCACATGAGCGAC CTCATGAGGCGCTCGCGCTTCCCGTTCGACACGAGCAACGAGATGTTGGCGGCACCCAGC AGCCACGACGACGACGACTTCTTCCGGGACCTCCCAGTGCTGGCGCGCGAGCAACAACCA GCGGCACAGCAACGCGAGAAGGGAGCACAGCCATCGCCAGTGGACAAGCAGACGGTCGCA GCAACCAATACCACCCGGGACAATGACCCGAATAAACAGACCAACGAGAACGATCATGTC CACCGTCGCGCCTTCTCGTCCTACTCCTTCAGCAACTCCTCCGTCGTGGACGACAAAGGC CGACGTGTGACGAGCACGCGCCGTCGCTACGAAGACTCGACCGGTCGCCTTAAAGCCGTG CACGAGCGCCAGGTCGAGGGCAAGACGCTCCGCACGACGTGGAACCGCGCGGGCCCCGAC GATAAGGGCCAGCACGAGTCCGTGTGTTCGAGCGGCTCGCCCGACGAGTTCGAGGCGCTG TGGCAGCAGACGCCCTTCGGTGCAGCACAGAAGAAGACCATCAAGCAGCAGCAGCAGCAG CAGCAGCAGTTGGAGCAAGGACAGCAGAGTCAACAGGAGAAGTCCCCAACCAGCTCGAAA CCGGAGACGGAGGCGACGCCAATGCAAGAGTGATGTTCATCGCACGTTCAGCAGATGATG GACGGCAGTAACGCAAGCAGCCAACGCGAAGCCTTGTCGCCCATTTGCTATCGACTGCGG GGTGAAACTTCCCAGTACCGCCAACTGCGTGAAAATGTGCAAACACGACGAAGGAGCCGA TACGAGGGTGAAGCTCAGCAACGTACACAAGTCGTTCGTCGTTCTACATACATGAGTAAA GAGTAATAAACAACTATTTAGTTTCCAAACCAC

Exercise IV
1. Do in silico PCR in FASTPCR for identifying the PCR product size and binding

sites.

2. Paste the target sequence and paste the primers click the run button find the PCR product size. Primers:
> ACGGAGCCTCCTTTTTCTTC > TGTCCTCATGGGAGCACATA

Seq:

>Phytophthora
CCTTGGCCTCTGCCTAATCACACAGATTCTAACAGGATTATTTCTCGCAATACACTACACAGCTGACA TCTCAACAGCCTTCTCCTCCGTCGCCCACATCTGTCGAGATGTTAACTACGGATGACTAATTCGAAAC ATTCATGCAAACGGAGCCTCCTTTTTCTTCATCTGCCTCTACCTTCACGTAGCCCGAGGCATATACTA TGGCTCATACCTCTACAAAGAAACCTGAAACATCGGAGTAGTTCTCCTACTCCTAACTATAATAACCG CCTTCGTAGGATATGTGCTCCCATGAGGACAGATATCCTTCTGAGGAGCCACCGTAATTACCAACCTT CTTTCCGCCTTCCCCTACATCGGGGACACCCTAGTACAATGAATCTGAGGTGGTTTCTCAGTAGACAA CGCCACCCTAACC

>Ralstonia
CCTAGGCCTCTGCCTTATTACACAAATCTTAACAGGACTATTTCTTGCAATACACTACACAGCTGACA TTTCAACAGCCTTCTCCTCCGTCGCCCACATCTGCCGAGACGTAAACTACGGGTGACTAATCCGAAAC GTCCACGCAAATGGCGCCTCCTTCTTCTTTATCTGCTTGTACCTTCACGTCGCACGAGGTATATACTA CGGCTCCTACCTCCAAAAAGAAACCTGAAACATCGGAGTAGTCCTCTTACTCCTCACCATAATAACCG CCTTCGTAGGCTATGTACTGCCCTGAGGACAAATATCATTTTGAGGGGCAACCGTAATCACTAACCTC CTTTCCGCCTTCCCGTACATCGGCGACACATTAGTGCAATGAATCTGAGGCGGCTTTTCAGTC