Design PCR amplification primers for directional cloning of

Design PCR amplification primers for directional cloning of GOI into pUC19 plasmid (map provided). Please show steps, and thinking process.

For primer design you have to run restriction analysis of your GOI and select suitable directional cloning sites in pUC19 multiple cloning site (MCS). Both selected sites should only cut once in pUC19 and should be absent in your GOI.

-Design forward and reverse primers for entire coding sequence of GOI.

-Match melting and PCR annealing temperature for both primers before introduction of RE sites.

-Introduce selected RE sites to forward and reverse primers.

-Add extension (“junk”) sequence to facilitate enzyme restriction digest. (Check selected enzymes’ cutting efficiency close to the ends of DNA fragment)

-Analyse primer self-complementarity and primer dimer formation for full primer sequences.

Gene of interest for cloning into pUC19:

  1 ATGGGGAGAC AGCCATGCTG TGACAAGCTA GGGGTGAAGA AAGGGCCGTG
51 GACGGTGGAG GAAGATAAGA AGCTTATAAA CTTCATACTA ACCAATGGCC
101 ATTGTTGCTG GCGTGCTTTG CCGAAGCTGG CCGGTCTCCG TCGCTGTGGA
151 AAGAGCTGCC GCCTCCGGTG GACTAACTAT CTCCGGCCTG ACTTAAAACG
201 AGGCCTTCTC TCGCATGATG AAGAACAACT TGTCATAGAT CTTCATGCTA
251 ATCTCGGCAA TAAGTGGTCT AAGATAGCTT CAAGATTACC TGGAAGAACA
301 GATAACGAAA TAAAAAACCA TTGGAATACT CATATCAAGA AGAAACTTCT
351 TAAGATGGGA ATCGATCCTA TGACCCATCA ACCCCTAAAT CAAGAACCTT
401 CTAATATCGA TAATTCCAAA ACCATTCCGT CCAATCCAGA CGATGTCTCA
451 GTGGAACCAA AGACAACTAA CACGAAATAC GTGGAGATAA GTGTCACGAC
501 AACAGAAGAA GAAAGTAGTA GCACGGTTAC TGATCAAAAC AGTTCGATGG
551 ATAATGAAAA TCATCTAATT GACAACATTT ATGATGATGA TGAATTGTTT
601 AGTTACTTAT GGTCCGACGA AACTACTAAA GATGAGGCCT CTTGGAGTGA
651 TAGTAACTTT GGTGTTGGTG GAACATTATA TGACCACAAT ATCTCCGGCG
701 CCGATGCAGA TTTTCCGATA TGGTCACCGG AAAGAATCAA TGACGAGAAG
751 ATGTTTTTGG ATTATTGTCA AGACTTTGGT GTTCATGATT TTGGGTTTTG
801 A

Eco0109 287 Pol 46 179 Ndel 183 Ethel 23s Bcgl 2215 Scal 21 Tsol 2 pUC18/19 gull 683 2686 bp NmeAll 1822 Psci 806 Cfr10I Eco31I 1 Eam1105l 1217 Multiple cloning sites of pUC18 5 GTAA AAC: GAC GGC CAG TGC CAA GCT TGC ATG CCT GCA GGT CGA CTC TAG A 3\' c ATT TTG CTG cce GTC ACG GTT CGA ACG TAC GGA CGT CCA GCT GAG ATC T Lacz Val Val Ala Leu Ala Leu Ser Ala His Arg Cys Thr Ser Glu Leu Ec136ll GG ATC CCC GGG TAC CGA GCT CGA ATT CGT MAT CAT GGT CAT AGC TGT TTC CTG 3 CC TAG GGG CCC ATG GCT CGA GCT TAA GCA TTA GTA CCA GTA. TCG ACA AAG GAC 5 Pro Asp Gly Pro Val Ser Ser Ser Asn Thr Ile Met Thr Met Multiple cloning sites of pUC19 5\' G TAA AAC GAC GGC CAG TGA ATT CGA GCT TAC CCG GGG ATC TAG 3\' c ATT TTG CTG cce GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG GAG ATC T Lacz Val Val Ala Leu Ser Asn Ser Ser Pro Val Arg Pro Asp Glu Leu GT CGA CCT GCA GGC ATG CAA GCT TGG cGT AAT CAT GGT CAT AGC TGT TTC CTG 3 CA GCT GGA CGT CCG TAC GTT CGA AOC GCA TTA GTA CCA GTA TCG ACA AAG GAC Ser Arg Cys Ala Ser Pro Thr lle Met Thr Met

Solution

Forward primer           GTGTTCATGATTTTGGGTTTTGA3’        from [801] [GOI]

                                    3’CACAAGTACTAAAACCCAAAACT5’             [primer]

Tm = 4(G+C) +2(A+T) = 4(1+7)+ 2(12+3) =4x8+ 2x15=62⁰C

Reverse primer                5’ATGGGGAGACAGCCATGCT    from [1] [GOI]

                                    3’TACCCCTCTGTCGGTACGA5’              [primer]

Tm = 4(G+C) +2(A+T) = 4(4+7)+ 2(3+6) =4x11+ 2x9=62⁰C

Introduce sacI or others which has 5’GCT3’ recognition site

Forward primer CACAAGTACTAAAACCCAAAACTTCG5’

Annealing temperature 62 + 4x2 +2=72⁰C

Same for reverse primer

Extension for Forward primer CACAAGTACTAAAACCCAAAACTTCGTCG5’

Extension for Reverse primer TACCCCTCTGTCGGTACGATCGTCG5’