Bcanton:Primers

Tools

 * Oligo analysis
 * Austin's clipboard

Template
Project - Forward homology sequence - 5'--3' Reverse homology sequence - 5'--3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'--3' Reverse primer - 5'--3' Notes -

BioBrick prefixes and suffixes

 * BioBrick prefix - 5'-GAATTC GCGGCCGC T TCTAGA G-3'
 * BioBrick prefix for coding regions - 5'-GAATTC GCGGCCGC T TCTAG-3'
 * BioBrick suffix - 5'-TACTAGT A GCGGCCG CTGCAG-3'
 * BioBrick suffix reverse complement - 5'-CTGCAG CGGCCGC T ACTAGTA-3'

11/8/05
T7 gene 1 - To PCR T7 gene 1 from the T7 genome with BioBrick ends Forward homology sequence - ATG AAC ACG ATT AAC ATC GCT AAG AAC GAC TTC Reverse homology sequence - 5'-TTA CGC GAA CGC GAA GTC CG-3' I want to order it ready to cut X/P so that I can ligate downstream of a promoter-RBS Prefix - 5'-CGC T TCTAG-3' Suffix - 5'-TAC TAG TAG CGG CCG CTG CAG CTG G-3' Suffix reverse complement - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA-3' T71F - 5'-CGCTTCTAG ATG AAC ACG ATT AAC ATC GCT AAG AAC GAC TTC-3' TM1 - 60C TM2 - 63C T71R - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA TTA CGC GAA CGC GAA GTC CG-3' TM1 - 60C TM2 - 72C

C0011 - ccdB - Biobricking the ccdB gene from E. coli. Sequence from pDEST14(Invitrogen) courtesy of Heather Forward homology sequence - 5'-ATG CAG TTT AAG GTT TAC ACC TAT AAA AGA GAG -3' Tm - 57C Reverse homology sequence - 5'-CCA TTA ACC TGA TGT TCT GGG GAA TAT AA-3' Reverse homology sequence reverse complement - 5'-TTA TAT TCC CCA GAA CAT CAG GTT AAT GG-3' Tm - 56.5C 5' additions - Will be cutting X/P Prefix - 5'-CGC T TCTAG-3' Suffix - 5'-TAC TAG TAG CGG CCG CTG CAG CTG G-3' Suffix reverse complement - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA-3' C0011F - 5'-CGC T TCTAG ATG CAG TTT AAG GTT TAC ACC TAT AAA AGA GAG-3' Tm - 61.4C C0011R - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA TTA TAT TCC CCA GAA CAT CAG GTT AAT GG-3' Tm 69.1C Notes - Just the usual biobrick homodimers

T7 Gene 5.3 - C7053 - Forward homology sequence - 5'-ATG AAC GAA AGA CAC TTA ACA GGT GC-3' Tm = 57.6C Reverse homology sequence - 5'-GGC AAG AGA AAC AAA GGC ATA AAA CTA TAG-3' Reverse homology sequence reverse complement - 5'-CTA TAG TTT TAT GCC TTT GTT TCT CTT GCC -3' Tm - 56.4C 5' additions Prefix - 5'-CGC T TCTAG-3' GTTTCTT C GAATTC GCGGCCGC T Suffix - 5'-TAC TAG TAG CGG CCG CTG CAG CTG G-3' Suffix reverse complement - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA-3' T75.3F - 5'-CGC T TCTAG ATG AAC GAA AGA CAC TTA ACA GGT GC-3' Tm - 62.6C T75.3R - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA CTA TAG TTT TAT GCC TTT GTT TCT CTT GCC-3' Tm- 69.2C Notes - T75.3F2 - 5'- GTTTCTT C GAATTC GCGGCCGC T TCTAG ATG AAC GAA AGA CAC TTA ACA GGT GC-3' Tm - 62.6C

T7 Gene 7.7 - C7077 - Forward homology sequence - 5'-GTG GAA GAC TGC ATT GAA TGG AC-3' Tm = 56.3C Reverse homology sequence - 5'-CAG CGT AAG ACA TGG AGA CAC ATT TA-3' I'm missing an A here. Reverse homology sequence should be - 5'-CAG CGT AAG ACA TGG AGA CAC ATT TAA-3' Reverse homology sequence reverse complement - 5'-TAA ATG TGT CTC CAT GTC TTA CGC TG-3' Tm - 56.8C This should be Reverse homology sequence reverse complement - 5'-TTAA ATG TGT CTC CAT GTC TTA CGC TG-3' Tm - 56.8C 5' additions Prefix - 5'-CGC T TCTAG-3' Suffix - 5'-TAC TAG TAG CGG CCG CTG CAG CTG G-3' Suffix reverse complement - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA-3' T77.7F - 5'-CGC T TCTAG GTG GAA GAC TGC ATT GAA TGG AC-3' Tm - 63.2C T77.7R - 5'-C CAG CTG CAG CGG CCG CTA CTA GTA TAA ATG TGT CTC CAT GTC TTA CGC TG-3' Tm - 69.7C This should be - T77.7R -    5'-C CAG CTG CAG CGG CCG CTA CTA GTA T TAA ATG TGT CTC CAT GTC TTA CGC TG-3' Tm - 69.7C Notes - T77.7F2 - 5'-GTTTCTT C GAATTC GCGGCCGC T TCTAG GTG GAA GAC TGC ATT GAA TGG AC-3' Tm - 63.2C R2182 - Forward homology sequence - 5'-cgcttctagagtaatacgtctcactatagggagatactagagtcacacaggaaagtactagtagcggccgctgcagCTGG-3' Reverse homology sequence - 3'-CTTAAGCGCCGGCGAAGATCTCATTATGCAGAGTGATATCCCTCTATGATCTCAGTGTGTCCTTTCATGATCATCGCCGGCGACGTCGACC-5' Reverse homology sequence reverse complement - 5'-CCAGCTGCAGCGGCCGCTACTAGTACTTTCCTGTGTGACTCTAGTATCTCCCTATAGTGAGACGTATTACTCTAGAAGCGGCCGCGAATTC-3' 5' additions - Already ready to be cut X/P Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' R2182F - 5'--3' R2182R - 5'--3' Notes -

R6182 - Forward homology sequence - 5'-gaattcgcggccgcttctagagtaatacgtctcactatagggagatactagaggtgtgtactagtagcggccgctgcagCTGG-3' Reverse homology sequence - 3'-CTTAAGCGCCGGCGAAGATCTCATTATGCAGAGTGATATCCCTCTATGATCTCCACACATGATCATCGCCGGCGACGTCGACC-5' Reverse homology sequence reverse complement - 5'-CCAGCTGCAGCGGCCGCTACTAGTACACACCTCTAGTATCTCCCTATAGTGAGACGTATTACTCTAGAAGCGGCCGCGAATTC-3' 5' additions - Already ready to be cut X/P Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' R6182F - 5'--3' R6182R - 5'--3' Notes -

PCR conditions

 * C0011 - 51C & 56C
 * C7053 - 51C & 57C
 * C7077 - 51C & 57/58C
 * Gene 1 - 55C 58C

12/6/05
Project - B0037 Forward homology sequence - 5'-aattcgcggccgcttctagaggtgtgtctagta-3' Reverse homology sequence - 5'-CTAGTACTAGACACACCTCTAGAAGCGGCCGCG-3' Reverse homology sequence reverse complement - 5'--3' 5' additions already cut E/S Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'-aattcgcggccgcttctagaggtgtgtctagta-3' Reverse primer - 5'-CTAGTACTAGACACACCTCTAGAAGCGGCCGCG-3' Notes -

Project - R8237 Forward homology sequence - 5'-aattcgcggccgcttctagagtaatacgtctcactatagggagatactagaggtgtgtctagta-3' Reverse homology sequence - 5'-CTAGTACTAGACACACCTCTAGTATCTCCCTATAGTGAGACGTATTACTCTAGAAGCGGCCGCG-3' Reverse homology sequence reverse complement - 5'--3' 5' additions already cut E/S Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'-aattcgcggccgcttctagagtaatacgtctcactatagggagatactagaggtgtgtctagta-3' Reverse primer - 5'-CTAGTACTAGACACACCTCTAGTATCTCCCTATAGTGAGACGTATTACTCTAGAAGCGGCCGCG-3' Notes -

Project - R4037 Forward homology sequence - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagaggtgtgtctagta-3' Reverse homology sequence - 5'-CTAGTACTAGACACACCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagaggtgtgtctagta-3' Reverse primer - 5'-CTAGTACTAGACACACCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Notes -

1/26/06
Project - E71101 Forward homology sequence - 5'-TAC TAG ATG CGT AAA GGA GAA GAA CTT TTC -3' Tm = 56.4C Reverse homology sequence - 5'--3' Reverse homology sequence reverse complement - 5'-CTG CAG CGG CCG CTA CTA G-3' Tm = 60.9C 5' additions Prefix - 5'-cctttctagagtaatacgtctcactatagggagatactagaggtgtgtctag-3' (add an XbaI site and the promoter and RBS) Suffix - 5'-GTT TCT CC-3' (adds random bases and extra bases to allow PstI to cut) Suffix reverse complement - 5'--3' E71101F - 5'-cctttctagagtaatacgtctcactatagggagatactagaggtgtgtctagTAC TAG ATG CGT AAA GGA GAA GAA CTT TTC-3' Tm = 67.1C E71101R - 5'-GTT TCT CCCTG CAG CGG CCG CTA CTA G-3' Tm = 66.1C Notes -

Project - E70101 Forward homology sequence - 5'-TAC TAG ATG CGT AAA GGA GAA GAA CTT TTC -3' Tm = 56.4C (same as above) Reverse homology sequence - 5'--3' Reverse homology sequence reverse complement - 5'-CTG CAG CGG CCG CTA CTA G-3' Tm = 60.9C 5' additions Prefix - 5'-cctttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagaggtgtgtctag-3' (add an XbaI site and the promoter and RBS) Suffix - 5'-GTT TCT CC-3' (adds random bases and extra bases to allow PstI to cut) Suffix reverse complement - 5'--3' E70101F - 5'-cctttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagaggtgtgtctagTAC TAG ATG CGT AAA GGA GAA GAA CTT TTC-3' Tm = 69.0C ( E70101R - 5'-GTT TCT CCCTG CAG CGG CCG CTA CTA G-3' Tm = 66.1C Notes - this is 113 bases and I can't reduce it by 13 bases. So I should get E71101 sorted and then just PCR R0040 onto that, which would save me having to include B0037

3/3/06
Project - E70102-annealing Forward homology sequence - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagtctcacgtgtgtcaagta-3' Reverse homology sequence - 5'-CTAGTActtgacacacgtgagaCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagtctcacgtgtgtcaagta-3' Reverse primer - 5'-CTAGTActtgacacacgtgagaCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Notes - I put this together by just copying R4037 from above and then replacing with the B0070 sequences. Its 101 bases so I can't order it.

Project - E70103-annealing Forward homology sequence - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagtctcacgtgtgtta-3' Reverse homology sequence - 5'-CTAGTAacacacgtgagaCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'-aattcgcggccgcttctagagtccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagtctcacgtgtgtta-3' Reverse primer - 5'-CTAGTAacacacgtgagaCTCTAGTAGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTATCACTGATAGGGACTCTAGAAGCGGCCGCG-3' Notes -

3/20/06
Project - E71102 Forward homology sequence - 5'-gtttctccgaattcgcggccgcttctagag taatacgtctcactatagggaga tactagagtctcacgtgtgtcaagta-3' Reverse homology sequence - 5'- TACTTGACACACGTGAGACTCTAGTATCTCCCTATAGTGAGACGTATTACTCTAGAAGCGGCCGCGAATTCGGAGAAAC -3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'--3' Reverse primer - 5'--3' 5'-gtttctccgaattcgcggccgcttctagag taatacgtctcactatagggaga tactagagtctcacgtgtgtcaagta-3' 3'-CAAAGAGGCTTAAGCGCCGGCGAAGATCTC ATTATGCAGAGTGATATCCCTCT ATGATCTCAGAGTGCACACAGTTCAT-5' 5'-   ctccgaattcgcggccgcttctagag taatacgtctcactatagggaga tac-3' 3'-                               GCAGAGTGATATCCCTCT ATGATCTCAGAGTGCACACAGTTCATGATCATCGC-5' 5'-CGCTACTAGTACTTGACA CACGTGAGACTCTAGTATCTCCCTATAGTGAGACG -E71102Rnew GCAGAGTGATATCCCTCT ATGATCTCAGAGTGCACACAGTTCATCGCTACTAT 5'- TATCATCGCTACTTGACACACGTGAGACTCTAGTATCTCCCTATAGTGAGACG -3' Notes -

Project - E71104 Forward homology sequence - 5'-gtttctccgaattcgcggccgcttctagag tcatacgactcactatagggaga tactagagtctcacgtgtgtcaagta-3' Reverse homology sequence - 5'--3' Reverse homology sequence reverse complement - 5'--3' 5' additions Prefix - 5'--3' Suffix - 5'--3' Suffix reverse complement - 5'--3' Forward primer - 5'--3' Reverse primer - 5'--3' Notes - 5'-gtttctccgaattcgcggccgcttctagag tcatacgactcactatagggaga tactagagtctcacgtgtgtcaagta-3' 3'-CAAAGAGGCTTAAGCGCCGGCGAAGATCTC AgTATGCTGAGTGATATCCCTCT ATGATCTCAGAGTGCACACAGTTCAT-5' 5'-ctccgaattcgcggccgcttctagag tcatacgactcactatagggaga tac-3' 3'-                               GCTGAGTGATATCCCTCT ATGATCTCAGAGTGCACACAGTTCATGATCATCGC-5' 5'-CGCTACTAGTACTTGACACACGTGAGACTCTAGTATCTCCCTATAGTGAGTCG-3' -E71104Rnew 5'- TATCATCGCTACTTGACACACGTGAGACTCTAGTATCTCCCTATAGTGAGTCG -3' So the reverse primer is the same except for one base:)