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BIGEASY LINEAR CLONING KIT

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1. CTGACCGCGATTGATACAGTCTTTCAGCAAATTAATTAACGACATCCTGTTTCCTCTCAAACATGCCCTTATC GTG TTCATCA AC ACG AAAGCAAAGCAACATAAAAAAAGCAAAGTGACTTAGAAAACGCAAAGTTAAGGTTCAAATCAA GATGCGC ACAGAAGCTA AGCTTCATCTAAGCGCAACGGTATTACTTACGTTGGTATATTTAAAACCTAACTTAATGA AAATGATAATAAA TCATACCAATTGCTATCAAAAGTTAAGCGAACATGCTGATTTTCACGCTGTTTATACACTTTGAGGCATCTCTATCTCTTCCGTCTCTA ATTGAAACACAATCAAAGAACATCAATCCATGTGACATCCCCCACTATCTAAGAACACCATAACAGAACACAACATAGGAATGCAACAT AATGTATCAATAATTCGGAACATATGCACTATATCATATCTCAATTACGGAACATATCAGCACACAATTGCCCATTATACGC Appendix H Recommended conditions for High throughput sequencing of pJAZZ clones Use the Millipore Montage 96 well kit to prep the DNA Use Phenix 384 well FrameStar PCR plates to set up the reactions do the ethanol ppt and run the sequencing samples For sequencing reactions use 3ul of template 60ng 180ng rxn 3ul of the following master mix Master mix for 100rxns 16 5 ul Big Dye V3 1 0 78 ul primer 320 pmol ul 2 5 pmol rxn 118 0 ul 5X ABI sequencing buffer 164 72 ul DIUF water 300 0 ul total Cycle 95 C for 4 min then 25 cycles of 95 C for 15 sec 55 C for 5 sec 60 C for 2 min Hold at 4C until ready for use Cleanup with ethanol precipitation or Sephadex G 50 Protocol courtesy of Laboratory for Genomics and Bioinformatic
2. eren at nenne KK KA KA raa KK KA KK KK KA 15 Appendix B Application Guide kk kk or KK KK KK KK KK KK KK KK KK KK KAKA iav uri DIS 15 Appendix C Abbreviated Protocol e celo a kk kk A KK KK KK er ek M KK KK en 16 Appendix D Vector Map Cloning Site and Sequencing Primers 17 Appendix E Troubleshooting Guide UA DU IU DUE IU UNIO DU IU DEPE 18 Appendix F Sequence of pJAZZ OC VECHON ccccccccccccceceeeceeeeeeeeeeeeeeeeeeeeeeeeeeneeeeeess 19 Appendix G Sequence of pJAZZ OK vector ccccccccccccccecceeeeceeeececeeeeeeceeeeeeeeeeeeesess 22 Appendix H Conditions for HTS of pJAZZ clones 24 Lucigen Corporation 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Kit Designations The BigEasy v2 0 Linear Cloning Kits are available with either a blunt or a Notl digestion of the pJAZZ OC or the pJAZZ OK vector The catalog numbers are listed below Please refer to Appendix B Application Guide for more information and recommended uses Lucigen s cloning kits Catalog numbers of Kits pJAZZ OC Blunt pJAZZ OC Noti pJAZZ OKBlunt pJAZZ OK Notl 43018 1 43024 1 43036 1 43042 1 43018 2 43024 2 43036 2 43042 2 43018 3 43024 3 43036 3 43042 3 Components amp Storage Cond
3. d 0 001 No UV 30s 60s 120s 120s 302nm 302nm 302nm 360nm Figure 2 Relative cloning efficiency of pUC19 after exposure to short or long wavelength UV light Intact pUC19 DNA was transformed after no UV exposure No UV or exposure to 302 nm UV light for 30 60 or 90 seconds 30s 302nm 60s 302nm 120s 302nm or to 360 nm UV light for 120 seconds 120s 360nm Cloning efficiencies were calculated relative to un irradiated pUC19 DNA To safely gel isolate large fragments we recommend running a duplicate lane of the insert DNA Stain photograph and physically mark the band in the duplicate lane place it next to an unstained lane containing Lucigen Corporation 7 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits the desired sample and excise the region of the unmarked gel corresponding to the position of the desired band This method completely avoids exposure of the band to ethidium bromide and UV light Materials and Equipment Needed The BigEasy v2 0 Linear Cloning Kits supply most of the items needed to efficiently generate recombinant clones While simple and convenient successful use of the BigEasy Kit requires proper planning for each step Please read the entire manual and prepare the necessary equipment and materials before starting Following ligation the following items are required for transformation e Electroporation apparatus and 0 1 cm cu
4. T GGATC GATA CCGCAGT GCC AGCAGATCGCGCCGT r CGCGGAAG ATGCAGA CCGGCGTGA ACTGCT CGAT CACT GCTGCCA C TATCTCA CCAGC GCATGG AACGCCACC CGTCGACA CCAGCTGCAGCTCCT TTCGATG CCCAGCGGACCAGC CGGTAATCGG TCAAACAGGCGCACT TCCGGCAACGCGA TATGGCCGGGA AAGCGTAGG TTTTCAGGC GGGCC GCTCA ACAGGGTGCG GTTCACGGGC ATCAAC CATCGTG CGGCCTGG ACGTAAA CC TACCGA GCTG CATGCA AGTTAG CGTAGG GGCGCA TGATGCAAA CCACGCCGAG GCTGGCA AATCATTTC CGGTCAC CG CAAAGACAACG GGCCGATGCC CTG CGCGTTTGA CCGGATCGG GCATCTGGA CTGAAG ATATCGACGG G G CGGTGG TTCTGGT GGCG CGCTGGGGA GCTG AACGCAGAGAAAC AGATGAGGC CACG CCACTTCGC ACCGCGAAGC CGCAGAACAT G CAGGAG GCAAGCGCG CGAGCGCGGCGGCTT CAGCAGCCCGGAG GCCGAAA ATCGACAC CAGG CGGTGGC CATA TGGCGC CCATAAGCTGC CAA GGCGCGACC GA AGCCATCA A r GGCCAGCCG ATTTTGAGC T GCATGAA CGAGCAAGGCCT GAAG GCG CGAAACCGA AATG ATTCC GAGCTGTACGG CGC GGCGGTG GAAG GTTTCTCC CAATCGA ACAC CGTCATA CCAGCATGA TCG GCTTG GG CGCG CATCG CCAG GTTG CAGCCAGGI TGTCAG CGCCGCG CGAACGTCGA l CAACAAG
5. CCCGTTG TTTCCCA ACCCCTCCGG TTATTTCGCG AC GATAGCAGG CCGCCTCCAT IC GG Lucigen Corporation 888 575 9695 608 831 9011 www lucigen com GCTGGCAACGCGT CCATAAA C CGGTCA CCGCCCACGCCT GTACAGCGAGGCGAACGT CCGGTACCGCCACCGGGA ATATCGC MAOSS3 v2 2 CGTGGCTG ACCTTGACCACATAAGGGTCGTAGCCCTCC CACGGC GCCGCTGGCTTAGAAACGCTTTCAGCAGCC C CGCGATGCTGGCCACTGGCCACAGGCGTA AGCCTCCAGTGCCTGGATAATTACTG 19 BigEasy v2 0 Linear Cloning Kits ATTG GGGGCGTCCGGAACG GCTCTG TGGATCGAG TGG CAATGG GGTCTG TTCCACC ACG GCGTGAATTG ATAAACCGG GGTTACCATGTATA CTATA CCTCGCGGCGCTTC CCACGATA AACGCATCG GGGG CGTGCATCGC CGTCTCTGG CAGGCGC CTTC GCC C GCAGG GCAGCG ATCTCTGGC GCG CTGTG GAAGCCGCCGA CGCGTCTGGTCTTAC GGATAGCCC CATG AGGAACT TAGATCCAAATCGCGATCCAC r CGACCGAGTCCGGGT TCGATGG CATA GACTCCAGGA GCGTAAAACG G GAGGGCG CGCCGA GAGG GTGTAGAAAC CCATGTCTG CTTCACCT CACGCATGC CTATC ATTTAG CCTGGAACTC GCG CGGCCTGT TAAAG GAATCAACGC CGCCG CCAGGG AGACCA CGAAATAGAA CACGG CCCGCG CATGGCCG CAAGGCGACGGGCGG AG CCAGGG CAACAGCAGGGG GACGAAC
6. tonA bla Amp SOpAB telN antA e BigEasy TSA Electrocompetent Cells are provided with supercoiled control pKanR plasmid DNA at a concentration of 1 ng ul The pKanR control plasmid is kanamycin resistant e For highest transformation efficiency use the provided Recovery Medium to resuspend the cells after electroporation Use of TB SOC or other media may result in lower transformation efficiencies Purification and Size Fractionation of DNA DNA must be purified from restriction or repair enzymes before ligation to pJAZZ vectors Agarose gel electrophoresis which is commonly used to size fractionate DNA fragments is sufficient for purification If the insert DNA is not fractionated by electrophoresis after repair or digestion it must be purified by binding to a DNA purification column or by phenol chloroform extraction to remove the repair enzymes Sensitivity of DNA to Short Wavelength UV Light DNA resolved on agarose gels is generally stained with ethidium bromide and visualized by illumination with ultraviolet light Exposure to short wavelength ultraviolet light e g 254 302 or 312 nm can reduce cloning efficiencies by several orders of magnitude Figure 2 Note that the wavelength of most UV transilluminators even those designated specifically for DNA visualization is typically 302 nm or 312 nm and can cause significant damage to DNA 10096 ooo r 10 hl r RHRHR cRH 1 moo ooo x 0 1 V 0 01
7. Sheared DNA Because the pJAZZ vectors have minimal cloning bias and can maintain large inserts they are ideal for random shotgun cloning of fragments up to 30 kb This process typically entails a fragmentation step to randomly shear the DNA an end repair step to generate blunt ends and a fractionation step to size select the fragments Mechanical methods of DNA fragmentation e g nebulization sonication hydrodynamic shearing are often preferred over enzymatic methods as they are more random and reduce cloning bias 5 For random shearing Lucigen recommends using the HydroShear instrument by Genomic Solutions formerly GeneMachines Fragments generated by the HydroShear device are repaired more efficiently than those produced through sonication or nebulization It aleo generates a tight distribution of fragments in a desired size range increasing the amount of DNA available for cloning 5 The shearing results are also highly reproducible Mechanical fragmentation results in a heterogeneous mix of blunt and 3 and 5 overhanging ends that may not ligate efficiently Successful library construction requires a robust repair method to convert these ragged ends to blunt ends The DNATerminator End Repair Kit is included in the BigEasy Kit to ensure maximal efficiency of blunt cloning see below C DNATerminator Kit for End Repair of Fragments for Blunt Cloning Lucigen s DNATerminator End Repair Kit Cat 40035 1 and 40035 2
8. CAGTGCGTA AACGGTGTTCCGT C CCA G CGGAAGAGAGA CCAGGC GCCAG CACGGGC CTC ACGCAGGCA ATCCGGTCC GC GGTCTCTG CAGCGATAG CGTACCAATAACAGG CCCGATC CCAGCGACGGCA CGA GACTCACGAACGGCAGGAA CATAACGGGC TGG GCACAAA AA CGTTGAAT CCAGGA AACGTC CCCGGCTG CGCTC G GTTGGCAGG AACGCGGTC GGAT CACG TCCTACAGC CTGCCAC TTGCTTCAG CGTCAG CAGCTC CGC CAGA TCAGTGCCA ACAGTAT CACGTTCTAA GT TTGGACGCC TAAACGGCG GCTT CAGTCG CGCCTCGTGTT CATACC CTTAATCA A CCCCA CAATGG CAGCGCA CTGCAAGC GGG AAA GAAACCCGAAGAAC GGGGAGA AAGGAAGGGCAT G ATCTC A AGCTGGCTA TAAT ATAAATT GTGCGC CATTTCGGC CCACC TAACAGG CTACGACAA GGATCCTC CTAACT AAGCCG GTGGCCAG GCTTAGC C CAGT AGCGGGACAGGT ACGCTGGAG ETCC CACTAGTG CGACTGGAA GATG GGAAAAAGCAGCGGT ATACCTAGC TAA TCACTTA GGCG GGG CG TGA GACACCCGGCGCAGTCTAT ATAATA AGCGATATAGAGGTC AGACGCAGAAAGGCCCACCCGAAGGI TGTCTAGGGCCCAATGGCCCGGGAGGCC CGGCCGCGACAAC CGTACCGCG ACAA GAG ATTAAAGAG GATT CACCTCT CCAGTGTGA ACA GCGGC AAG AC AGCGGGCAGTGAGCG CAACGCAAT AATG TGAGTTAGC CAC CATTAGGC ACCC
9. CATGGCCG CAAGGCGACGGGCGG AG CCAGGG CAACAGCAGGGG GACGAAC T GGATC GATA CCGCAGT GCC AGCAGATCGCGCCGT r CGCGGAAG ATGCAGA CCGGCGTGA ACTGCT CGAT CACT GCTGCCA C TATCTCA CCAGC GCATGG AACGCCACC CGTCGACA CCAGCTGCAGCTCCT TTCGATG CCCAGCGGACCAGC CGGTAATCGG TCAAACAGGCGCACT TCCGGCAACGCGA TATGGCCGGGA AAGCGTAGG TTTTCAGGC GGGCC GCTCA ACAGGGTGCG GTTCACGGGC ATCAAC CATCGTG CGGCCTGG ACGTAAA CC TACCGA GCTG CATGCA AGTTAG CGTAGG GGCGCA TGATGCAAA CCACGCCGAG GCTGGCA AATCATTTC CGGTCAC CG CAAAGACAACG GGCCGATGCC CTG CGCGTTTGA CCGGATCGG GCATCTGGA CTGAAG ATATCGACGG G G CGGTGG TTCTGGT GGCG CGCTGGGGA GCTG AACGCAGAGAAAC AGATGAGGC CACG CCACTTCGC ACCGCGAAGC CGCAGAACAT G CAGGAG GCAAGCGCG CGAGCGCGGCGGCTT CAGCAGCCCGGAG GCCGAAA ATCGACAC CAGG CGGTGGC CATA TGGCGC CCATAAGCTGC CAA GGCGCGACC GA AGCCATCA A r GGCCAGCCG ATTTTGAGC T GCATGAA CGAGCAAGGCCT GAAG GCG CGAAACCGA AATG ATTCC GAGCTGTACGG CGC GGCGGTG GAAG GTTTCTCC CAATCGA ACAC CGTCATA CCAGCATGA TCG GCTTG GG CGCG CATCG CCAG
10. CC GAAA GAAAAGACGGAGAGAGCCTTCATTGCG CTTCC TCGTC ACGGCT GCAAA CCAGCAGGCCC r GGCGAACT A CAGCGTGGCC CATAACTGG AGATAG GC GG GCG GGGCG r GGCGACGAGC AACTGAA CTGG CACC CTCGCTC GGATGA GC AATGG CGCTGAC GAG GAGCAGAGCCCACAAGCGC CATGCGA CGCGCGCGACCTGCAGAAGTTCCGCAG CAACCTGCAGCAGGCGTTCCTCAA ACGGGCTGACAGAACGAGGACAAA CGGGA GGAAAGGCGGTGGA AGCTGCAAAT G AATTTGAACA CCAGACAGGG GTAAC C GGTATGCA CAACACCGGT ACAAGAAGAAACCGGCCCAACCGAAG CCTATC T GTATAGGG GCTACCACCAGAG GGCCCCAT TAACACACAAAAAAACACGCTGGCGCG CTACCGCCAAAGCAGAACGCACG GCT TACTGAC CCATACGGCCACGAAT GAGCCACT GATCAGAACC G GTGCGC C GTCA CGGGGTTGAGAGGCCCGGC ATGGGATTTTTTGTCCGTGCGGACGAC GAGAATCTCTATAGGGGTGGTAGC CTGAGCCACCATAATTCAGGTATGCGCAGA GCAGA GCTGCAGCGGG CAA AATT AGG GGATA ACCCCGTGACCAG GATCAG GGAACGGAA CATCACGACG CACCACAAAGAAAG C CATCCA TCGG ATTGTCGA ATTGGAG AG C CAGCAGGI CGGTAATC ATTGATGACCGCAGCCACCT TACAGCGGAACGAACCACAAACGG CACGTGCACAGGTGTTTTTATAGT AGATGTTGTCTCAAACC CAGACGCTGCCAGAACGTCG CGACCTG ACTTATC GCTTCCAG G CGAACCC AAGCG A C G
11. GAAATTGAGCTCGACGAGGGTGGCGGC C CAAGCCTGCAAAAAATAACGGGGACGGA AATCGG GGCTT GGACA CGCCG CCCGAC AT GCAGGGAC GAGGCC GTCCCTACCCATCCCCTGCAAGGGACG CCCCGCGTAAAGCGGGGCTTAAATTCGG A CTGCAA CGCTGGC GATGTTA GGCAGCACC C CCAGTTCCG AGCAGC CCCG CG A AGACACGCA CAATATCG GT GTGA GCCCAGAGCCGT TCATCCACGG A GGGTTCGG AAAC GTGC A GTCG A GATGGGTTC CAGGCCCCGAAGTTCT ACAAAAGCATAC CGCGGCAGCGGAGGA AAACCGAGGGTCAA AGCGCCACAAACCACGGGI ACCGCG TCGG CGCCG CCGGA ATGGTG C G CGGCAGC CGTGGA e A CTGCAGCCG GCAGGCGCGGCGGAGAGCCAT AGCTGGCAGAACC AGAGACCAGA TC CGTTCGTCA CCACAAGACA GCCGCCAG GCGTTTCC AGCTTTTCAATGGCCAGCTCAAAATGTGCT ITAATATC rCCAG AC CCGGCGACCGCCACGAACTACATCGCGC CGGCGCAGCTCGGCGATGATTGCCGGGAGA ACGCCGCTAACCCATGCGTTACGGTACTGA CCAG CCCAC G GGTTCACCGGCGTTCTTAGGCTCAGGCTCG CGGAACCGCCAGGC CGTCCCCTGTTTC G CCGATACCA CCTCCCCGGAT I CAGCTGC TCATCATGA TCCAGC l CACCACCGACAAGAACCACCCG CAGAAAGGGACAGGGAGCAGCCGCGAGCTTCC TACGCAA GCAT CATTTAC CAGAAGGG CCCTGGCCATCCGATCAAGTT GTGCC GGCTATATTCAATGCAGCACAGATATCC ATAGGGTGGC
12. GAGC CGGGT AGGCCC CCAGAA GAAAAAA GAA GAAAACGGATCGC lr CCGCAGGAGCAGCCACAA C ITA GCCAAAGC GGCTCTTC CAGCACACGCAGGCCAG CAACGAAG GCAGAGC GA AACGCG AACGAAAGGA CCGGGGAGGAA ACCGGCCA GTTG TTCA GC CATTCCGTTG CTG CG AACAAAGCAT CG CACGCGACG TCCCAGGCGCGATGGAAAG CGGAGGCGGCG TTCTGGTCG GCACCGA C TETTO ATCAC A GCTTCAG CACGGCGA AC CCGAAAACA AACTGA GCCATCC TGCTCTGC CAA A CGCCAG G GTAG GTGGTATAGC GC CTACACCAC CTCA GTCAGCTCA CACG GCCGCGT GCTCCGCG TAAACG AGAAT CACTA C GTAAGG CTGAAGGACG CA A GCC CCA GCGAAT AGCATTTTT ATC GAGTT GCCGAC C GGGTTTTT CGTCT TCGGCTGCTACGG CTGG GCG CAACCCCGACAAAG ATCGTGGC AC CCTGAAAA ATA GA CGGATTAAA ATAGTCAG CAA AAACCCTG ATTGTA CA CTACCCTCAACCA GAACGA G ATCG ACCGACTAC GAAGA CAC A CA GTGA CTCGCCGTTA TA GGATAACCCG CACAAACCAG GTAG CGCTGAATC GG GAGAG ACATCC C AGCACTATCAAGG AG AATGCTGC CG ACCCGGTACAAGCG AAGTGAAGAA CCCGACCG CAGTGCGTA AACGGTGTTCCGT C CCA G CGGAAGA
13. GATAAGGAG AGTGTGACGAAAGCAGCATAA AACAT TAAGCGCAGAACAATA GTA CCGGTGTTGTGTTCCTTTGTTATTCTG TAA GAGCAAGG AAAAA CGG GAGTTGA C AAAGCCGCAGCCGCACGG TAACCGCGAA AAAAATA AATAAA A CGGAAAAG ACT AAC GCCTATA ATCC CACATG GCAAAAAT CATCG AGCAAGACT ACAA TATC A CTAAGCCC AAG A CTAAAT AATAAAAAAA CCAACAAGGC GAAAGAA ACAGCGAAGAAT CGTCACTTG C GTGTTACGATATCCAGAGACTTAGAAAC AACACGCTTG ATAAGAACG GAA AGAGGCAATTGATGCCTCAGACCGCCCA CGTTA GAGCAGGGCAAGAAAGCGG AATGATAAAAGAAAG GATGATAAA TCCGTGGGAAAGGA ACATCATAGCTTTGA AT ATAA GCCGC GCCGAAG CTTCA GGC TCTATGCCTACGGCTAATATTCGCCAG AGTTA CAAA GTAAGAATAGGCTCTAAAGGCAGTGA ATCCA GA CTGCG GCGGAGCGTACATC ATACAGCAA TTG CGAT GGAGTT AGAAGAACT GGGCCGA GCTCTITA TACACC C G GTGAT TAAACAG GATGATTGGAAGGAGCGCCGTGAC AGC CAAGG GCGCGAGAAGAAGCG CAACCATGAGGTTCTGTACCATCTGCAG AATGTTGTGGTTATTGACTACCCA ACATACA GCAG CTATCTATGA GCTCTGGC GGGCAAGCT GCGG TAAAAAACGC A CAGGGCGAAGAA A GAA AA CCTGCGAC TTA A G AAAC ACTCGTTCTGGAATGGCACC GGCCTTT GA C GAAGA AAAAGC GAGA GT AA
14. GTA CCGGTGTTGTGTTCCTTTGTTATTCTG TAA GAGCAAGG AAAAA CGG GAGTTGA C AAAGCCGCAGCCGCACGG TAACCGCGAA AAAAATA AATAAA A CGGAAAAG ACT AAC GCCTATA ATCC CACATG GCAAAAAT CATCG AGCAAGACT ACAA TATC A CTAAGCCC AAG A CTAAAT AATAAAAAAA CCAACAAGGC GAAAGAA ACAGCGAAGAAT CGTCACTTG C GTGTTACGATATCCAGAGACTTAGAAAC AACACGCTTG ATAAGAACG GAA AGAGGCAATTGATGCCTCAGACCGCCCA CGTTA GAGCAGGGCAAGAAAGCGG AATGATAAAAGAAAG GATGATAAA TCCGTGGGAAAGGA ACATCATAGCTTTGA AT ATAA GCCGC GCCGAAG CTTCA GGC TCTATGCCTACGGCTAATATTCGCCAG AGTTA CAAA GTAAGAATAGGCTCTAAAGGCAGTGA ATCCA GA CTGCG GCGGAGCGTACATC ATACAGCAA TTG CGAT GGAGTT AGAAGAACT GGGCCGA GCTCTITA TACACC C G GTGAT TAAACAG GATGATTGGAAGGAGCGCCGTGAC AGC CAAGG GCGCGAGAAGAAGCG CAACCATGAGGTTCTGTACCATCTGCAG AATGTTGTGGTTATTGACTACCCA ACATACA GCAG CTATCTATGA GCTCTGGC GGGCAAGCT GCGG TAAAAAACGC A CAGGGCGAAGAA A GAA AA CCTGCGAC TTA A G AAAC ACTCGTTCTGGAATGGCACC GGCCTTT GA C GAAGA AAAAGC GAGA GT AA G TAACCAGAACGA CAGGGTG AATTTGCC ATA C
15. GTTG CAGCCAGGI TGTCAG CGCCGCG CGAACGTCGA l CAACAAGCA GCACTGCAG TTTCACCATCAC AGGCTGG GGTG AAATCGTCGA GGAACTGTAGTC CAGC GCATGG AACG C CGGCGAACGTCGAAC CGATAACCTCGG CCGGGAG CAATCACGTGAA CA GATCCGA GCAGCCAGGC TTTC AGGT CGGTA GATCGTTGG AAGCG GTTGAATAC GCC ACCGGG AGTCA GATAGC GG GC GGCGCATCAGCGGTTGCCAGCAGCC CCATCGCAC CATCGGCA GCCCGGCCAGAAT CCACCA GGCACGCAGGCCC AATGGCATG CTC CAG G GTTGCT AAG ATGGAGTTGATGC AAATAGTCAG CTTG CGGGA CCTGA GCGCGACGGAAACGCCACGCGTGGAC AA GCAGAATG CGGC CG CGCC GCAGCA CAG GGACGCAGGAGGC CCCTTCGG CA CTG CA CGA C TAACGA GCCCTGCAG CTG GCGGCGTATG GAAGGT CGGGTTGGT GATG CGTTCAC GAACGAACG ICATCACC CATCGGTCATCC G TAACGG ACCG ATAAA CTGCGATAC GGCATG GCGTGA CAGCA GCCAA CGACAACAGAT A CG CAACGGCGC GTTGCACGGC CCATATCCC ATG GCGCGGCAC GCAGCGTGCTGC GAAGCAG GGAGG GAAAAGGCGCA CAAA ATGC ACCC GGCATA AAAA l AGGCCCAGCTCCT ACGCGCCAGCGCGT CATCGAAGCCGAAGA GGCGCCAT ACGCGGAT CCTTCTCC G TAG CGCGTCGCT CGA CCT AGGCCGCC GACCCCA A CAGGGA LrCCCGGCGCGGAC
16. provides an efficient and convenient method for end repairing DNA fragments The insert DNA needs to be relatively free of RNA before end repairing Even moderate amounts of contaminating RNA will severely impair the efficiency of the end repair reaction resulting in poor cloning results We recommend the use of RNase I which breaks RNA down into nucleotides to remove residual RNA associated with DNA purification RNase DNase free is available from Lucigen Cat 30104 1 and 30104 2 We do NOT recommend use of RNase A because it is a site specific endonuclease that will not degrade the RNA sufficiently DNATerminator End Repair Reaction The DNATerminator End Repair Kit has been optimized for processing approximately 0 2 15 pmol of DNA fragments equivalent to 1 10 ug of DNA fragmented to 5 kb Buffers used for fragmentation of the DNA must be removed before beginning the DNATerminator reaction Ammonium ions strongly interfere with the end repair reaction so they must be removed prior to the reaction The most common sources of ammonium ions are PCR buffer and ammonium acetate used for ethanol precipitation Fragments should be purified by binding to a DNA purification column or precipitation with ethanol and sodium acetate Mix the following components in a microfuge tube y ul fragmented DNA in water 10 ul 5X DNATerminator End Repair Buffer 2 ul DNATerminator End Repair Enzymes X ul H2O 50 ul final volume Incubate 30 minu
17. ATGC GAATTGCG C AATGCTA TAGCAAAAGCA GCTCTGC GCA CTGA C GATGAGG T GTTAAAGGA A GGAAAGGA GTTTCAGGAAAGTATACGGT CTCA GAAGCAAAATTATTCGTTGAATTATTAACA GATACAAGGTCTGAGAACGGCAGGATA AA TAACCC GG G AAATCA TTCGGCG GCTCGCA GACGA CGC GAGAACACCCAGC ATGAGATG C TCCGCGTC AGGCTGG CTGG GGC C GCAC ATAAGCAG GA CCACGG TTCAAGC TGCAGAAAC TTTGCCGC GAATCCG GAT GA GAGACCAT G CA GGACGA GGAGCAGGACCCATCAGCAAAAAT GGGGCAG l CGACGAACCGGA GAACCAACCGAAGAGGAAGGGCCAGAAGAACAT GAA TCG ATGCCAG GAT ACGTACAAGATAGAG GAAACG GAAGGAT AC GCTGGCCAACCC ACAGC TAGGCGGAAAC GAATACGATGGAAAGCAT AAAAGAAAAGCCACCGGTG GCAGC GCAACTAC GAG ra GC TAACCAACAGCACTCT GGCGAGAACGGGCAGTGGCAGC CCCAAGACGACGAGC AAAACCCG GGTCCGGCCCCGCCGATAGCCCTATGGCCGCAATGCGATCCGCATGG CAGCCAAC rGcC 1 GGCCAACTTC GGAAAAACG GCCAGAG A GACCG lr CGACGAGGA CCAGAACCTGGCGACC CGTG ATAAAGATAGCCGCGCTATTTAC G TCTTCATGGAGATTCTCGGACAC GAAGTTGGGGATGAAAACACC G GTGACGC rCCGGGCC AAAT GCTC GAAGA AGAGACACC GGCGTCCGTCTCCATGAAACCGTTAAGCAG AGCCCGACGATGATTAGCCGGTACCTGGAG GCAATCGTCCTGCCTGATGAA GGATGAAGA
18. G TAACCAGAACGA CAGGGTG AATTTGCC ATA C ATGC GAATTGCG C AATGCTA TAGCAAAAGCA GCTCTGC GCA CTGA C GATGAGG T GTTAAAGGA A GGAAAGGA GTTTCAGGAAAGTATACGGT CTCA GAAGCAAAATTATTCGTTGAATTATTAACA GATACAAGGTCTGAGAACGGCAGGATA AA TAACCC GG G AAATCA TTCGGCG GCTCGCA GACGA CGC GAGAACACCCAGC ATGAGATG C TCCGCGTC AGGCTGG CTGG GGC C GCAC ATAAGCAG GA CCACGG TTCAAGC TGCAGAAAC TTTGCCGC GAATCCG GAT GA GAGACCAT G CA GGACGA GGAGCAGGACCCATCAGCAAAAAT GGGGCAG l CGACGAACCGGA GAACCAACCGAAGAGGAAGGGCCAGAAGAACAT GAA TCG ATGCCAG GAT ACGTACAAGATAGAG GAAACG GAAGGAT AC GCTGGCCAACCC ACAGC TAGGCGGAAAC GAATACGATGGAAAGCAT AAAAGAAAAGCCACCGGTG GCAGC GCAACTAC GAG ra GC TAACCAACAGCACTCT GGCGAGAACGGGCAGTGGCAGC CCCAAGACGACGAGC AAAACCCG GGTCCGGCCCCGCCGATAGCCCTATGGCCGCAATGCGATCCGCATGG CAGCCAAC rGcC 1 GGCCAACTTC GGAAAAACG GCCAGAG A GACCG lr CGACGAGGA CCAGAACCTGGCGACC CGTG ATAAAGATAGCCGCGCTATTTAC G TCTTCATGGAGATTCTCGGACAC GAAGTTGGGGATGAAAACACC G GTGACGC rCCGGGCC AAAT GCTC GAAGA AGAGACACC GGCGTCCGTCTCCATGAAACCGTTAAGCAG AGCCCGACGATGATTAGC
19. antibiotic For the pKanR control use YT kanamycin 30 pg ml Incubate overnight at 37 C oo N O O WO N Colony Growth 1 Pick white colonies at random and grow in TB medium containing the appropriate antibiotic plus 1X Arabinose Induction Solution if desired Lucigen Corporation 16 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Appendix D Vector Map Cloning Site and Sequencing Primers The pJAZZ OC or OK vector is supplied predigested at either Smal blunt or Notl sites with dephosphorylated ends The sequences of the SL1 and NZ RevC primers are as follows SL1 5 CAGTCCAGTTACGCTGGAGTC NZ RevC 5 AAATGGTCAGTTAATCAGTTCT The GenBank accession number for the pJAZZ OC vector is EF583812 The GenBank accession number for the pJAZZ OK vector is not yet available SL1 Primer Notl Ahdl Apal Smal Insert CAGTCCAGTTACGCTGGAGTCACTAGTGCGGCCGCGACAACTTGTCTAGGGCCCAATGGCCC GTCAGGTCAATGCGACCTCAGTGATCACGCCGGCGCTGTTGAACAGATCCCGGGTTACCGGG Insert Smal Ahdl Notl a Ao 5 CCCGGTAATCTGAACTTCAGTTCGCCGGCGATGTTGACCTGGAACGACCATGTATCTTGACTAATTGACTGGTAAA lt ______________________ NZ RevC Primer Lucigen Corporation 17 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Appendix E Troubleshooting Guide Problem Probable Cause Solution Very few or no Inef
20. components or derivatives of the product 4 use of this product or materials made therefrom to provide a service information or data e g DNA sequence to a third party in return for a fee or other consideration or 5 resale of the product or its components or derivatives whether or not such product or its components or derivatives are resold for use in research Lucigen Corporation will not assert a claim of infringement against the buyer of this product provided that none of this product or any of its components or any claim in the foregoing patent or patent applications was used in the manufacture of a product for commercial purposes Academic Not for Profit and For Profit institutions must obtain a separate license from Lucigen Corporation to use this product for any purpose other than those permitted above It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties If the purchaser is not willing to accept these use limitations Lucigen Corporation is willing to accept return of the product for a full refund For information on obtaining a license to use this product for purposes other than those permitted above contact Lucigen Corporation 2120 W Greenview Dr Middleton WI 53562 Email Lucigen lucigen com Phone 608 831 9011 Fax 608 831 9012 Lucigen Corporation 2 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kit
21. number is desired add Arabinose Induction Solution to the cultures 1 pl per ml of culture final concentration 0 01 arabinose Table 1 Plating Transformed Cells Reaction Plate Drug Resistance ul Plate Experimental Insert 500 ng per ligation Cam or Kan 20 amp 100 Control Insert Positive Control 500 ng ul Cam or Kan 50 No Insert Control Vector Background Cam or Kan 50 pKanR Transformation Control Plasmid Kanamycin 2 1 ul diluted 1 100 to 10 pg ul Expected Results The results presented below are expected when cloning 500 ng of intact purified DNA fragments with Blunt or Notl ends and 5 phosphate groups into Lucigen s BigEasy TSA Electrocompetent Cells The background number of empty pJAZZ vectors is constant lt 25 colonies per 50 ul of cells plated unless kinase or repair enzymes are introduced as contaminants Two types of background colonies are possible 1 Blue colonies are produced from the trace amounts of undigested vector supplied in the pJAZZ preparation and 2 White colonies with no inserts may arise from ligation of the vector arms The total number of recombinant clones is typically 100 fold greater than the background of white colonies from self ligated pJAZZ vector Table 2 Expected Transformation Results from Electroporation Reaction CFU Ligation Efficiency pJAZZ Blunt plus Blunt Control Insert gt 100 000 gt 95 inserts pJAZZ Notl vector
22. 10 minutes at 70 C 4 Purify DNA by binding to matrix phenol chloroform extraction or gel electrophoresis Elute in deionized water Do NOT use 256 302 or 312 nm UV light to visualize the DNA Ligation 1 Briefly centrifuge and gently mix the BigEasy pJAZZ Vector 2 Briefly centrifuge and gently mix the CloneDirect Buffer 3 Combine the following components in a 1 5 ml tube Add ligase last x ul Insert DNA 100 500 ng 5 phosphorylated proper termini 1 0 2 0 ul pJAZZ OC or OK Vector 1 0 ul 10X CloneDirect Ligation buffer contains ATP 1 0 ul CloneSmart DNA Ligase 2 U ul y ul H2O 10 0 ul total reaction volume 4 Incubate 2 hours at room temperature 5 ESSENTIAL Heat denature the ligation reaction 15 minutes at 70 C 6 Cool 15 seconds at room temperature and 15 seconds on ice Spin 1 minute at 12 000 rpm The ligation reaction can be used directly for electroporation without further purification Electroporation Have Recovery Medium at room temperature for transformations Chill electroporation cuvettes on ice Thaw BigEasy TSA Electrocompetent Cells on wet ice Add 1 ul of heat treated ligation reaction to the cells on ice Pipet 25 ul of the cell DNA mixture to a chilled electroporation cuvette Electroporate Immediately add 975 ul of room temperature Recovery Medium Place in culture tube Shake at 250 rpm for 1 hour at 37 C Spread up to 100 ul per plate on YT agar plates containing the appropriate
23. A TAAGCACAAG ab CAGTCAG TGCTCAATG T TATCCGGCCT ATTCA GAAAGACGG GAAAACC GAAACGTT CA CGCTC GGAG GAA GAGC ACCACGACGA GG GATA CCGGCAGTT GGGATAG G TCACCCTTG GGCC A GAGTT CACCAGT GATTTAAACGTGGCCAA A GGACAAC ese AAAGGGT TA ICT TGAGAA A G H CGCCCCCGT CAAGGTGC GA CTGCGATGAG CAATGCGG CCAGG CCC GACA GTG GCGG CGA AAAA G G GGCAA CAACGAGTAGGACGCGG AACGGAAAGTACAACCGGG AGCCAGCGCGACG ACGCCGA AGCGCGCG GCCGCTGGCGA GGCAGGGCGGGGCGTAACC GAAGGGCCAGGCAGC CA GGTTCA AGG CA GACAGAAGT G GG GGATTATG CCG GGAAGCAGTGTA CG CTGCG GCC C GAA GCAAGGCTAACCA G GCAAA GTGA CAAAAGCC CGAGACCCGGCCAGCA CAGGAACG GGCTTCCA CCGG G CACCA CGGCAGAA CGGAGGC GGGCAAATA A CTACACA CGTCTCAGCCAATCCCTGGGT ACGCAAGGCGA ATATTC GCT AATGAA ACAACAGTA T GAC TTC GCTAGATCTG T GTTGG TTATCGCA A TCAGCGT G CGCGTTTAC AACC G GGTACA AGA GCAGTCCC GCGCCTGG GTAC e ACTCGGCCCC T ATCG CA GGTCTTTGG CA c CAGGTC GCAAC CATTT CC CTG GACCGCGA A CAAAA CAGGAGCCGGI AC
24. AA ACGC CCG GGCA GCG ATTC GTAAGTGA AACCGGCGCG G GTGACGGGC AA GTGATCTT C GGCAAAAGTGC ICAGATGA TTCTCGTTAAC CGT CGGTGC TCACCA CCAGCC AAAA CGCTGCA TTCC AGCTCAGGG CATCC CAAAAG TGGGTAAACAC GGAGGCC GCTGC AGATGGAT G GTTG CGACAACGTGAA r GCCCCGGT CC TCCT CAGCTGAGG CATG GGCGAAAC TGTTAGCGG A GCGGTCGTTA GTCTG GGCGAGCGACTTCCT GACGCCGGAAAAC CGAGAGCGCAAG ATT AAACCGCAATGAC CCTCTGCAG CGAAAT CACCGTTAG CGTCCA AAGCGCCAAATACGTCACGAA CCAACCATCCAAGCAGGCG GG GGGACAGAGG GATG GCGCTGGTC GGAACTTGC TCGCAATTTTT ACCGCG GCGCCCCC AAG CTTTCGGGC G CAGACGCGGGAGAGTACGCAGC TTGGTACCG TATTCA lr CAGCCCTCTCAGGC GTGCAGGT CTCCA G ATTT GA GCGCAGCCGGG GTCGAATC ACATGT CAAAGCGTTT CGGGT GCAG CACCATTCAGA ATCGCCATG AAGTTGTTG CCC TACCGCAT CGA ACG GTCG GCCAGCCAACAAT CCAA rcCGCC GTCCACGGCTCCA CGCACTGTC GTGA CTGG AAGGCCAGGAGCCA CTTCAGCTGG CGAA G TCCTGCTCCA CTGATTG CCTGGC GGAAT GCTCCA GGCGTG CGCGCG CAAGCA AATAGATT GCGTGCACG GAAAAGCCACTCCAGGAGCCAGCG CCACACGGCC CTTC CCGGCTGAG TTAAATCA
25. ARTGC vectors contain similar features but are optimized for cloning PCR products For cloning large inserts or very difficult DNAs including regions containing long stretches of di tri or tetra nucleotide repeats the BigEasy v2 0 Linear Cloning Kits are recommended The pSMART BAC vector in the CopyRight Cloning kits is useful for inserts up to 200 kb Use of the E clon 10G or BigEasy strains is essential for cloning inserts that may be methylated such as genomic DNA isolated directly from plant or mammalian cells as these strains contain the inactive mcr and mrr alleles mcrA mrr hsdRMS mcrBC Vector Insert DNA Source Desired Use Vector Name Copy Cosmid Genomic AT Rich Digestion Plasmid or cDNA Large Subcloning PCR etc BAC etc Difficult Sequencing pSMART HC Kan High IpSMART LC Kan Low IpSMARTGC HCK High IpSMARTGC LCK Low IpSMART BAC Single Mid pJAZZ OC or OK Low Mid Lucigen Corporation 15 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Appendix C Abbreviated Protocol Please see Manual for detailed instructions Insert DNA Preparation 1 Generate target DNA fragments by shearing restriction digestion or PCR 2 If necessary repair the DNA to generate blunt ends with 5 phosphate groups 3 Heat denature the repair reaction
26. ATCCGAATG ATAA CGCTA AGC GTCGCG CCCGTTG TTTCCCA ACCCCTCCGG TTATTTCGCG AC GATAGCAGG CCGCCTCCAT IC GG Lucigen Corporation 888 575 9695 608 831 9011 www lucigen com GCTGGCAACGCGT CCATAAA C CGGTCA CCGCCCACGCCT GTACAGCGAGGCGAACGT CCGGTACCGCCACCGGGA ATATCGC MAOSS3 v2 2 CGTGGCTG ACCTTGACCACATAAGGGTCGTAGCCCTCC CACGGC GCCGCTGGCTTAGAAACGCTTTCAGCAGCC C CGCGATGCTGGCCACTGGCCACAGGCGTA AGCCTCCAGTGCCTGGATAATTACTG 22 BigEasy v2 0 Linear Cloning Kits ATTG GGGGCGTCCGGAACG GCTCTG TGGATCGAG TGG CAATGG GGTCTG TTCCACC ACG GCGTGAATTG ATAAACCGG GGTTACCATGTATA CTATA CCTCGCGGCGCTTC CCACGATA AACGCATCG GGGG CGTGCATCGC CGTCTCTGG CAGGCGC CTTC GCC C GCAGG GCAGCG ATCTCTGGC GCG CTGTG GAAGCCGCCGA CGCGTCTGGTCTTAC GGATAGCCC CATG AGGAACT TAGATCCAAATCGCGATCCAC r CGACCGAGTCCGGGT TCGATGG CATA GACTCCAGGA GCGTAAAACG G GAGGGCG CGCCGA GAGG GTGTAGAAAC CCATGTCTG CTTCACCT CACGCATGC CTATC ATTTAG CCTGGAACTC GCG CGGCCTGT TAAAG GAATCAACGC CGCCG CCAGGG AGACCA CGAAATAGAA CACGG CCCGCG
27. B GEAsY V2 0 LINEAR CLONING KT IMPORTANT 80 C and 20 C Storage Required Immediately Upon Receipt Lucigen Corporation Advanced Products for Molecular Biology 2120 W Greenview Drive Middleton WI 53562 Toll Free 888 575 9695 Phone 608 831 9011 FAX 608 831 9012 Email lucigen 9 lucigen com www lucigen com FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE MAOSS v2 2 BigEasy v2 0 Linear Cloning Kits Technical Support Lucigen is dedicated to the success and satisfaction of our customers Our products are tested to assure they perform as specified when used according to our recommendations It is imperative that the reagents supplied by the user especially the DNA targets to be cloned are of the highest quality Please follow the manual carefully and contact our technical service representatives if additional information is necessary We encourage you to contact us with your comments regarding the performance of our products in your applications Thank you Notice of Limited Label License Copyright Patents Warranties Disclaimers and Trademarks Copyright 2007 by Lucigen Corp All rights reserved TSA and CloneDirect are trademarks of Lucigen Corp Lucigen BigEasy pJAZZ CloneSmart pSMART DNATerminator PCRTerminator and E cloni are registered trademarks of Lucigen Corp HydroShear and GeneMachines are registered trademarks of Genomic Solutions Corporation Ann Arbor MI Vent is a trademark of Ne
28. CA GCACTGCAG TTTCACCATCAC AGGCTGG GGTG AAATCGTCGA GGAACTGTAGTC CAGC GCATGG AACG C CGGCGAACGTCGAAC CGATAACCTCGG CCGGGAG CAATCACGTGAA CA GATCCGA GCAGCCAGGC TTTC AGGT CGGTA GATCGTTGG AAGCG GTTGAATAC GCC ACCGGG AGTCA GATAGC GG GC GGCGCATCAGCGGTTGCCAGCAGCC CCATCGCAC CATCGGCA GCCCGGCCAGAAT CCACCA GGCACGCAGGCCC AATGGCATG CTC CAG G GTTGCT AAG ATGGAGTTGATGC AAATAGTCAG CTTG CGGGA CCTGA GCGCGACGGAAACGCCACGCGTGGAC AA GCAGAATG CGGC CG CGCC GCAGCA CAG GGACGCAGGAGGC CCCTTCGG CA CTG CA CGA C TAACGA GCCCTGCAG CTG GCGGCGTATG GAAGGT CGGGTTGGT GATG CGTTCAC GAACGAACG ICATCACC CATCGGTCATCC G TAACGG ACCG ATAAA CTGCGATAC GGCATG GCGTGA CAGCA GCCAA CGACAACAGAT A CG CAACGGCGC GTTGCACGGC CCATATCCC ATG GCGCGGCAC GCAGCGTGCTGC GAAGCAG GGAGG GAAAAGGCGCA CAAA ATGC ACCC GGCATA AAAA l AGGCCCAGCTCCT ACGCGCCAGCGCGT CATCGAAGCCGAAGA GGCGCCAT ACGCGGAT CCTTCTCC G TAG CGCGTCGCT CGA CCT AGGCCGCC GACCCCA A CAGGGA LrCCCGGCGCGGACAA ACGC CCG GGCA GCG ATTC GTAAGTGA AACCGGCGC
29. CA GGC ACAC A GCTTCCGGC CGTATG GTGTGGAATTGTGAG CGGACAACAA CACACAGGAAACAGC ATGACCA TGATTA CGCCAAGCTA A GGTGAGACTA AGAATAC CAAGC GCATGCGATACG ATCG AAC GATGGA CCGACGCACGT GCGA ATTCGC CCTATAGTGA GTCG ATTACAA CACTGGCCG CGTTTTACAACG CGTGACTGGGAAAACCC GGCGTCACCCAAC AATC GCCTTG CAGCACATCC CCCT TCGCCAGC GGCGTAA AGCGAAGAGGCCCGCACCGA CGCCC CC CAACAG GCGCAGCTGAATGG CGAA CTTAAGTAGGCC CC CGGGCCATTA GACTTGAAG Lucigen Corporation 888 57 608 83 5 9695 1 9011 www lucigen com CAAGCGGCCGCT MAOSS3 v2 2 ACAAC GGACC GC TGGTACA AGAACTGA AACT 23 BigEasy v2 0 Linear Cloning Kits GACCATTTAAATCATACCAACATGGTCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCC CGTTTTAATCTGATCGGCACGTAAG AGGTTCCAAC CACCATAATGAAATAAGATCACTACCGGGCGTAT GAGTTATCGAGA CAGGAGCTAAGGAAGCTAAAATG AGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGA ATATGGGTATAAATGGGCTCGCGA AATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTG CTGAAACATGGCAAAGGTAGCG GCCAATGATGTTACAGATGAGATGGTCAGGCTAAACTGGCTGACGGAA ATGCCTCTTCCGACCATCAAGCA ATCCGTACTCC GATGATGCATGGTTACTCACCACTGCGATCCCAGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTG GATGCGCTGGCAGTGTTCCTGCGCCGGTTG
30. CATTCGATTCCTG GTAATTGTCCTTTTAACGGCGATCGCGTA CGTCTCGCTCAG GCGCAATCACGAATGAATAACGGTTTGGTTGGTGCGAGTGA GATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAA ATGCATAAAC GCCATTCTCACCGGATTCAGTCGTCACTCATGGTGA CTCACTTGATAACCTTA TGACGAGGGGAAATTA ATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCC CATTACAGAAACGGC ICAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAG CACTTGATGCTCGATGAGTT C AACCTAGGTGACAGAAGTCAAAAGCCTCCGGTCGGAGGCT GACTTTCTGCTAGATCTG CAATGCGGTGAAGGGCCAGGCAGCT GGGGATTATGTCGAGACCCGGCCAGCATGTTGG TATCGCATATTCAGCGTTGTCGCG ACCCAGGTAAAATGGAAGCAGTGTATC GTCTGCGTGAATGTGCAAATCAGGAACGTAACCGTGGTACATAGATGCAGTCCCTTGCGGGTCGTTCCCTTCAACGAGTATGACGCGGTG CCCTTGCAAGGCTAACCATTGCGCCTGGTGTACTGCAGATGAGG ATAAACCCCTCCCTTGTGTGACATAACGGAAAGTACAACCGG GTT ATCGTCAGGTCTTTGGTTTGGGTTACCAAACACACTCCGCATATGGCTAATTTGGTCAATTGTGTAGCCAGCGCGACGTTCTAC TCGGCCCCTCATCTCAAAATCAGGAGCCGGTAGACGACCAGCTT CCGCGTCTCTGATAGCCTGCGGTGTTACGCCGATCAGGTCTGC AACTTCTGTTATACCCCAGCGGCGAGTAATACGACGCGCTTCCGGGCTGTCATCGCCGAACTGTGCGATGGCAATAGCGCGCGTCATTTC
31. CCCAGCGGCGAGTAAT TGGG CCCTTATC GTG TTCA CA AC GTTCAAA CAA GATGCGC ACAGAAGC ACG GATACAG CT A AGCTTCATC AAGCGCAACG CTTAATGA AAATGATAA AAATCA ACCAA GCATCTCTA GAACACAACA CTTCCG CTCTA AT GAAACACAAT GC ATCAAAAG CAAAGAACA AGGAATGCAACAT AATTGCCCA ATACGC Lucigen Corporation 888 575 9695 608 831 9011 www lucigen com AA GTATCAA AA TCGGAACA AAGCGAACA TACGACGCGC CAGCAAATTAAT AAAGCAAAGCAACATAAAAAAAGCAAAG GCAGA ACCAAACACAC AGACGACCAGCTTT CCGGGC AACGACATCC GAGGT ITA AAACCCC GCGGG CCE CGT GT CCGCA ATGGC AAT TGGTCAA TCCGCG GTCA CTCTGATAGCCT CGCCGAAC GTG G Se GAC CTCAAACATG TAGAAAACGCAAAGTTAAG GTA AC ACG TGGTA ATTTAAAACCTAA GC GAT CAATCCATGTGACA CC CCCAC TCACGCTG TA ATC ATGCACTATA MAOSS3 v2 2 CA ATC CAAT ACGGAACA ACACTTTGAG AAGAACACCATAACA TATCAGCACAC 21 BigEasy v2 0 Linear Cloning Kits Appendix G Sequence of pJAZZ OK vector 13042 bp lacZ stuffer fragment is underlined GCGTATAA GGAC ATTGTGTGC CTATTATG TT TCTC GGGGGAACCGGGA CAAGGCGACAAAACGAAGAGAA GCAGAAAAGAAT TA
32. CCTGGCG TTGCTGCTTCCGAGTAGCAATCCTCT GAGCCGCT CCAGCTCATTCCTCCACAAAACAGGCA CCCATCCTCTGCGA AAATCA GA A GTC C AAA GCGTAGCAAA GGCACAAGAAA G CCGTTCCA CCC GTAATCC I GCCTGCTC GGA CAGCAAA ACCTTC AAGGC GTAGAAC GCAAAA CGCTCTCGTTCACATGCTGTACGTAGA GGAAAGATCG CCTTGAC CATA TAAACGCCTC Ce GTTCTTTC CCCCCAC GGCCA AATCTCAAG G CAGCAAA TGGGTCAGCC A GCCGTAG AA G CCGATTC AAAGC CCCACCG CATA C C CCACCATCAG AACTGCCCA ACAGTAC CAGC GTTCGTAA CCAG GC GCAGC G GA AAGCGGACCAGC CGGACACCGGCAGGCGGC AATAGG GA GCCACACACCAAAAACAGGAATCA CTCACGAAGAACCTTATCCAATAGCCCTGC CTTTTCGGC CA AGCAACCAGG CCATCCA CCACG CA TTGTAGATCATGCGCCACTATTCA GATTGAAAGGTCAAGCCAACCACGTCCAGA CATTCTAC GGCCATC CAGGAAC CTCTTCG GCCTCAA CGCCTTCATGTAGTCTTCTGTACGGAACCA AGCCCGGATGCGGTTATCGCACAGCTCGCG C GGTGTCGAC CTGGC ICGTC G GTGGTC ATCG ACGA GAA GGAAG l GACAAGGCA CCA CCCGGCCAG GCTCTTA CGC GGCAA GTCATAG GGCTTCACCTTATAGGCTTTTAGAAGCGCC GCAGCAA AAAC CCC CACTATCTGAGAACCCGTTCATCCGAATG ATAA CGCTA AGC GTCGCG
33. CGACCACGGTCGCAC CATGCGCT TAA GA GI GAGC CGGGT AGGCCC CCAGAA GAAAAAA GAA GAAAACGGATCGC lr CCGCAGGAGCAGCCACAA C ITA GCCAAAGC GGCTCTTC CAGCACACGCAGGCCAG CAACGAAG GCAGAGC GA AACGCG AACGAAAGGA CCGGGGAGGAA ACCGGCCA GTTG TTCA GC CATTCCGTTG CTG CG AACAAAGCAT CG CACGCGACG TCCCAGGCGCGATGGAAAG CGGAGGCGGCG TTCTGGTCG GCACCGA C TETTO ATCAC A GCTTCAG CACGGCGA AC CCGAAAACA AACTGA GCCATCC TGCTCTGC CAA A CGCCAG G GTAG GTGGTATAGC GC CTACACCAC CTCA GTCAGCTCA CACG GCCGCGT GCTCCGCG TAAACG AGAAT CACTA C GTAAGG CTGAAGGACG CA A GCC CCA GCGAAT AGCATTTTT ATC GAGTT GCCGAC C GGGTTTTT CGTCT TCGGCTGCTACGG CTGG GCG CAACCCCGACAAAG ATCGTGGC AC CCTGAAAA ATA GA CGGATTAAA ATAGTCAG CAA AAACCCTG ATTGTA CA CTACCCTCAACCA GAACGA G ATCG ACCGACTAC GAAGA CAC A CA GTGA CTCGCCGTTA TA GGATAACCCG CACAAACCAG GTAG CGCTGAATC GG GAGAG ACATCC C AGCACTATCAAGG AG AATGCTGC CG ACCCGGTACAAGCG AAGTGAAGAA CCCGACCG
34. CGGTACCTGGAG GCAATCGTCCTGCCTGATGAA GGATGAAGA GAAATTGAGCTCGACGAGGGTGGCGGC C CAAGCCTGCAAAAAATAACGGGGACGGA AATCGG GGCTT GGACA CGCCG CCCGAC AT GCAGGGAC GAGGCC GTCCCTACCCATCCCCTGCAAGGGACG CCCCGCGTAAAGCGGGGCTTAAATTCGG A CTGCAA CGCTGGC GATGTTA GGCAGCACC C CCAGTTCCG AGCAGC CCCG CG A AGACACGCA CAATATCG GT GTGA GCCCAGAGCCGT TCATCCACGG A GGGTTCGG AAAC GTGC A GTCG A GATGGGTTC CAGGCCCCGAAGTTCT ACAAAAGCATAC CGCGGCAGCGGAGGA AAACCGAGGGTCAA AGCGCCACAAACCACGGGI ACCGCG TCGG CGCCG CCGGA ATGGTG C G CGGCAGC CGTGGA e A CTGCAGCCG GCAGGCGCGGCGGAGAGCCAT AGCTGGCAGAACC AGAGACCAGA TC CGTTCGTCA CCACAAGACA GCCGCCAG GCGTTTCC AGCTTTTCAATGGCCAGCTCAAAATGTGCT ITAATATC rCCAG AC CCGGCGACCGCCACGAACTACATCGCGC CGGCGCAGCTCGGCGATGATTGCCGGGAGA ACGCCGCTAACCCATGCGTTACGGTACTGA CCAG CCCAC G GGTTCACCGGCGTTCTTAGGCTCAGGCTCG CGGAACCGCCAGGC CGTCCCCTGTTTC G CCGATACCA CCTCCCCGGAT I CAGCTGC TCATCATGA TCCAGC l CACCACCGACAAGAACCACCCG CAGAAAGGGACAGGGAGCAGCCGCGAGCTTCC TACGCAA GCAT CATTTAC CAGAAGGG CCCTGGCCATCCGATCAAGTT G
35. CTTATC GCTTCCAG G CGAACCC AAGCG A C G CCTGGCG TTGCTGCTTCCGAGTAGCAATCCTCT GAGCCGCT CCAGCTCATTCCTCCACAAAACAGGCA CCCATCCTCTGCGA AAATCA GA A GTC C AAA GCGTAGCAAA GGCACAAGAAA G CCGTTCCA CCC GTAATCC I GCCTGCTC GGA CAGCAAA ACCTTC AAGGC GTAGAAC GCAAAA CGCTCTCGTTCACATGCTGTACGTAGA GGAAAGATCG CCTTGAC CATA TAAACGCCTC Ce GTTCTTTC CCCCCAC GGCCA AATCTCAAG G CAGCAAA TGGGTCAGCC A GCCGTAG AA G CCGATTC AAAGC CCCACCG CATA C C CCACCATCAG AACTGCCCA ACAGTAC CAGC GTTCGTAA CCAG GC GCAGC G GA AAGCGGACCAGC CGGACACCGGCAGGCGGC AATAGG GA GCCACACACCAAAAACAGGAATCA CTCACGAAGAACCTTATCCAATAGCCCTGC CTTTTCGGC CA AGCAACCAGG CCATCCA CCACG CA TTGTAGATCATGCGCCACTATTCA GATTGAAAGGTCAAGCCAACCACGTCCAGA CATTCTAC GGCCATC CAGGAAC CTCTTCG GCCTCAA CGCCTTCATGTAGTCTTCTGTACGGAACCA AGCCCGGATGCGGTTATCGCACAGCTCGCG C GGTGTCGAC CTGGC ICGTC G GTGGTC ATCG ACGA GAA GGAAG l GACAAGGCA CCA CCCGGCCAG GCTCTTA CGC GGCAA GTCATAG GGCTTCACCTTATAGGCTTTTAGAAGCGCC GCAGCAA AAAC CCC CACTATCTGAGAACCCGTTC
36. G G GTGACGGGC AA GTGATCTT C GGCAAAAGTGC ICAGATGA TTCTCGTTAAC CGT CGGTGC TCACCA CCAGCC AAAA CGCTGCA TTCC AGCTCAGGG CATCC CAAAAG TGGGTAAACAC GGAGGCC GCTGC AGATGGAT G GTTG CGACAACGTGAA r GCCCCGGT CC TCCT CAGCTGAGG CATG GGCGAAAC TGTTAGCGG A GCGGTCGTTA GTCTG GGCGAGCGACTTCCT GACGCCGGAAAAC CGAGAGCGCAAG ATT AAACCGCAATGAC CCTCTGCAG CGAAAT CACCGTTAG CGTCCA AAGCGCCAAATACGTCACGAA CCAACCATCCAAGCAGGCG GG GGGACAGAGG GATG GCGCTGGTC GGAACTTGC TCGCAATTTTT ACCGCG GCGCCCCC AAG CTTTCGGGC G CAGACGCGGGAGAGTACGCAGC TTGGTACCG TATTCA lr CAGCCCTCTCAGGC GTGCAGGT CTCCA G ATTT GA GCGCAGCCGGG GTCGAATC ACATGT CAAAGCGTTT CGGGT GCAG CACCATTCAGA ATCGCCATG AAGTTGTTG CCC TACCGCAT CGA ACG GTCG GCCAGCCAACAAT CCAA rcCGCC GTCCACGGCTCCA CGCACTGTC GTGA CTGG AAGGCCAGGAGCCA CTTCAGCTGG CGAA G TCCTGCTCCA CTGATTG CCTGGC GGAAT GCTCCA GGCGTG CGCGCG CAAGCA AATAGATT GCGTGCACG GAAAAGCCACTCCAGGAGCCAGCG CCACACGGCC CTTC CCGGCTGAG TTAAATCA CGACCACGGTCGCAC CATGCGCT TAA GA GI
37. GAG CGGACAACAA CACACAGGAAACAGC ATGACCA TGATTA CGCCAAGCTA A GGTGAGACTA AGAATAC CAAGC GCATGCGATACG ATCG AAC GATGGA CCGACGCACGT GCGA ATTCGC CCTATAGTGA GTCG ATTACAA CACTGGCCG CGTTTTACAACG CGTGACTGGGAAAACCC GGCGTCACCCAAC AATC GCCTTG CAGCACATCC CCCT TCGCCAGC GGCGTAA AGCGAAGAGGCCCGCACCGA CGCCC CC CAACAG GCGCAGCTGAATGG CGAA CTTAAGTAGGCC CC CGGGCCATTA GACTTGAAG Lucigen Corporation 888 57 608 83 5 9695 1 9011 www lucigen com CAAGCGGCCGCT MAOSS3 v2 2 ACAAC GGACC GC TGGTACA AGAACTGA AACT 20 BigEasy v2 0 Linear Cloning Kits GACCAT TAAA CATACCAACATGGTCAAAT TAAAACGAAAGGC AGG TACCTA CATTCT CCAACT GGAGAAAAAAA AACCAGACCG GCCCGCC TCACCATAA CACTGGATA ACCACCG GAAATAAGATCAC AC CAG CGAAAGAC GGGCC CGTTT CGGGCGTAT TTTGAGT GATA A CCCAA CAGCT GGA ATTACGG Ce T GGCA AAAGACCGT CGTAAAGAA GATGAATGC TTACACCG xb CCATGAGCAAAC GCAAGATG GGCG GTTACGG CA CCGGAA C GTATGGCAA TAAAGAAAAAT ATCGAGA AATCTGATCGGCACGTAAG TTCAGGAGCTAAGGAAGCTAAAAT CA T GAGGC
38. GAGA CCAGGC GCCAG CACGGGC CTC ACGCAGGCA ATCCGGTCC GC GGTCTCTG CAGCGATAG CGTACCAATAACAGG CCCGATC CCAGCGACGGCA CGA GACTCACGAACGGCAGGAA CATAACGGGC TGG GCACAAA AA CGTTGAAT CCAGGA AACGTC CCCGGCTG CGCTC G GTTGGCAGG AACGCGGTC GGAT CACG TCCTACAGC CTGCCAC TTGCTTCAG CGTCAG CAGCTC CGC CAGA TCAGTGCCA ACAGTAT CACGTTCTAA GT TTGGACGCC TAAACGGCG GCTT CAGTCG CGCCTCGTGTT CATACC CTTAATCA A CCCCA CAATGG CAGCGCA CTGCAAGC GGG AAA GAAACCCGAAGAAC GGGGAGA AAGGAAGGGCAT G ATCTC A AGCTGGCTA TAAT ATAAATT GTGCGC CATTTCGGC CCACC TAACAGG CTACGACAA GGATCCTC CTAACT AAGCCG GTGGCCAG GCTTAGC C CAGT AGCGGGACAGGT ACGCTGGAG ETCC CACTAGTG CGACTGGAA GATG GGAAAAAGCAGCGGT ATACCTAGC TAA TCACTTA GGCG GGG CG TGA GACACCCGGCGCAGTCTAT ATAATA AGCGATATAGAGGTC AGACGCAGAAAGGCCCACCCGAAGGI TGTCTAGGGCCCAATGGCCCGGGAGGCC CGGCCGCGACAAC CGTACCGCG ACAA GAG ATTAAAGAG GATT CACCTCT CCAGTGTGA ACA GCGGC AAG AC AGCGGGCAGTGAGCG CAACGCAAT AATG TGAGTTAGC CAC CATTAGGC ACCCCA GGC ACAC A GCTTCCGGC CGTATG GTGTGGAATTGT
39. T4 PNK 10 ul Total Incubate at 37 C for 15 minutes Add 2 4 ul of this reaction directly to a 50 100 ul PCR mix and amplify Occasionally the kinase buffer interferes with the PCR reaction In these cases we recommend either ordering primers synthesized with 5 phosphates or kinasing the final PCR product see below Alternately blunt PCR products that lack 5 phosphate groups can be treated with T4 PNK or with the DNATerminator Kit included to add the phosphates The PCR products must first be purified to remove the PCR buffer as ammonium ions in the buffer strongly inhibit the phosphorylation reaction After phosphorylation the products must be purified again to remove the kinase activity Tailed PCR products PCR products created with non proofreading enzymes such as Taq or Tfl polymerases have single 3 single base overhangs that must be removed before ligation In addition they require addition of 5 phosphate groups To clone these PCR products we recommend using Lucigen s PCRTerminator End Repair Kit Cat 40037 1 which generates blunt 5 phosphorylated ends Lucigen Corporation 9 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits NOTE Ali PCR products should be purified by gel fractionation before cloning to remove PCR primers and spurious PCR products After gel isolation and purification of the desired fragment proceed with Ligation to the pJAZZ Vector p 10
40. TGCC GGCTATATTCAATGCAGCACAGATATCC ATAGGGTGGC CC GAAA GAAAAGACGGAGAGAGCCTTCATTGCG CTTCC TCGTC ACGGCT GCAAA CCAGCAGGCCC r GGCGAACT A CAGCGTGGCC CATAACTGG AGATAG GC GG GCG GGGCG r GGCGACGAGC AACTGAA CTGG CACC CTCGCTC GGATGA GC AATGG CGCTGAC GAG GAGCAGAGCCCACAAGCGC CATGCGA CGCGCGCGACCTGCAGAAGTTCCGCAG CAACCTGCAGCAGGCGTTCCTCAA ACGGGCTGACAGAACGAGGACAAA CGGGA GGAAAGGCGGTGGA AGCTGCAAAT G AATTTGAACA CCAGACAGGG GTAAC C GGTATGCA CAACACCGGT ACAAGAAGAAACCGGCCCAACCGAAG CCTATC T GTATAGGG GCTACCACCAGAG GGCCCCAT TAACACACAAAAAAACACGCTGGCGCG CTACCGCCAAAGCAGAACGCACG GCT TACTGAC CCATACGGCCACGAAT GAGCCACT GATCAGAACC G GTGCGC C GTCA CGGGGTTGAGAGGCCCGGC ATGGGATTTTTTGTCCGTGCGGACGAC GAGAATCTCTATAGGGGTGGTAGC CTGAGCCACCATAATTCAGGTATGCGCAGA GCAGA GCTGCAGCGGG CAA AATT AGG GGATA ACCCCGTGACCAG GATCAG GGAACGGAA CATCACGACG CACCACAAAGAAAG C CATCCA TCGG ATTGTCGA ATTGGAG AG C CAGCAGGI CGGTAATC ATTGATGACCGCAGCCACCT TACAGCGGAACGAACCACAAACGG CACGTGCACAGGTGTTTTTATAGT AGATGTTGTCTCAAACC CAGACGCTGCCAGAACGTCG CGACCTG A
41. a linear multicopy vector based on the mini replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures Nucleic Acids Res 27 613 2 Ravin NV Ravin VK 1998 Cloning of large imperfect palindromes in circular and linear vectors Genetika 34 38 44 3 Godiska et al Submitted 4 Godiska R Patterson M Schoenfeld T Mead DA 2005 Beyond pUC Vectors for Cloning Unstable DNA n DNA Sequencing Optimizing the Process and Analysis J Kieleczawa ed Jones and Bartlett Publishers Sudbury MA 5 Thorstenson YR Hunicke Smith SP Oefner PJ Davis RW 1998 An automated hydrodynamic process for controlled unbiased DNA shearing Genome Res 8 848 55 Lucigen Corporation 14 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Appendix A Media Recipes YT CXI and YT KXI Agar Medium for Plating of Transformants Add the YT Agar powder provided with the kit to 500 ml of deionized water Autoclave and cool to 55 C Add the appropriate filter sterilized antibiotic to the cooled medium e g 15 mg kanamycin 500 ml 30 ug ml final for KXI or 6 25 mg of chloramphenicol per 500ml 12 5 ug ml final for CXI Add XGAL to a final concentration of 20 mg L 20 ug ml and IPTG to 1 mM YT Agar is available to purchase separately as 5 packets with catalog number 60025 1 Temperatures of gt 55 C may destroy the antibiotics Do NOT add antibiotics to hot media Pour appro
42. e SOC or other media 2 Place electroporation cuvettes 0 1 cm gap on ice 3 Remove BigEasy TSA cells from the 80 C freezer and thaw completely on wet ice 10 15 minutes Lucigen Corporation 12 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits 4 Add 1 ul of the heat treated BigEasy v2 0 Ligation reaction to the 25 ul of cells on ice Failure to heat inactivate the ligation reaction will prevent transformation Stir briefly with pipet tip do not pipet up and down to mix which can introduce air bubbles and warm the cells Use of more than 2 ul of ligation mix may cause electrical arcing during electroporation 5 Carefully pipet 25 ul of the cell DNA mixture into a chilled electroporation cuvette without introducing bubbles Quickly flick the cuvette downward with your wrist to deposit the cells across the bottom of the well Electroporate according to the conditions recommended above 6 Within 10 seconds of the pulse add 975 ul of Recovery Medium to the cuvette and pipet up and down three times to resuspend the cells Transfer the cells and Recovery Medium to a culture tube 7 Place the tube in a shaking incubator at 250 rpm for 1 hour at 37 C 8 Spread up to 100 ul of transformed cells on YT CXI agar plates 9 Incubate the plates overnight at 37 C 10 Transformed clones can be further grown in TB or in any other rich culture medium with 12 5 ug ml chloramphenicol If higher copy
43. en Corporation 4 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Container 3 DNATerminator End Repair Kit provided with Blunt kits only Store at 20 C 5 Reactions 10 Reactions 20 Reactions DNATerminator End Repair Enzyme 20 ul 20 ul 40 ul DNATerminator 5X End Repair Buffer 100 ul 100 ul 200 ul Kit Description Lucigen s BigEasy v2 0 Linear Cloning Kits patent pending provide an unprecedented ability to maintain DNAs that are otherwise unclonable The BigEasy Kit is ideal for constructing shotgun libraries with large inserts or for cloning smaller products particularly when the target DNA is especially difficult to clone in conventional vectors The BigEasy Kit is based on the novel linear cloning plasmids pJAZZ Figure 1 Refs 1 3 which are not subject to supercoiling in the cell Conventional circular plasmids are maintained in multiple states of supercoiling by the action of DNA topoisomerase and gyrase Supercoiling induces torsional stress in the plasmid DNA which is associated with structural instability of sequences that are AT rich or contain inverted repeats 4 The ends of the pJAZZ vectors can rotate freely as the molecule is replicated so it is not under torsional stress As a result numerous classes of structure rich sequences are much more stable In addition the pJAZZ vectors incorporate Lucigen s patented CloneSmart technology for transcription
44. fficient for one transformation reaction each Transformation is carried out in a cuvette with a gap of 0 1 cm Optimal settings for electroporation are listed in the table below Typical time constants are 3 5 to 4 5 msec Optimal Setting Alternate Settings 20 5096 lower efficiencies 1 0 mm cuvette 1 0 mm cuvette 10 uF 25 uF 600 Ohms 200 Ohms 1800 Volts 1600 2000 Volts Suggested Electroporation Systems Bio Rad Micro Pulser 165 2100 Bio Rad E coli Pulser 165 2102 Bio Rad Gene Pulser Il 165 2105 BTX ECM630 Electroporation System Eppendorf Model 2510 Optional transformation control reactions include electroporation with 1 ul of a 1 100 dilution of the supplied supercoiled pKanR plasmid DNA 10 pg ul final concentration Transformation Protocol ESSENTIAL Ligation reactions must be heat denatured at 70 C for 15 minutes before transformation e Successful results are obtained with cuvettes from Eppendorf Cat 4307 000 569 BTX Model 610 or BioRad Cat 165 2089 Users have reported difficulties using Lucigen s electrocompetent cells with Invitrogen cuvettes Cat 65 0030 e The cells must be completely thawed on ice before use Electroporation cuvettes must be thoroughly pre chilled on ice before use 1 Have Recovery Medium and 17 mm x 100 mm sterile culture tubes readily available at room temperature one tube for each transformation reaction Transformation efficiency may decrease with the us
45. ficient end repair Check the insert DNA for self ligation by gel transformants electrophoresis Repeat end repair if necessary Contaminating enzymes in ligation reaction Heat denature end repair reaction or restriction digest 10 minutes at 70 C Purify DNA by extraction or adsorption to matrix No DNA degraded DNA or insufficient amount of DNA Check insert DNA by gel electrophoresis Determine concentration of insert and add the correct amount Use the supplied control insert to test the system Ligation reaction failed Check the insert DNA for self ligation by gel electrophoresis Repeat end repair if necessary Be sure insert DNA is phosphorylated Use the supplied control insert to test ligation reaction Inadequate heat denaturation of ligation reaction Be certain to heat denature for 15 min at 70 C Skipping this step may lower the number of transformants by 2 3 orders of magnitude Loss of DNA during precipitation DO NOT precipitate DNA after ligation reaction It is not necessary with this protocol and these cells Incorrect recovery media Use the Recovery Medium provided in the Kit for electrocompetent and chemically competent cells Improper electroporation conditions Use BTX or BioRad electroporation cuvettes with a gap of 0 1 cm Pre chill cuvettes on ice Add the 1 ul of DNA to 25 ul of pre aliquotted cells on wet ice DO NOT add the cells to the DNA Wrong ant
46. free cloning U S Pat 6 709 861 which further reduces instability or loss of insert DNA Large fragments or inserts with high AT content are cloned easily with this vector The BigEasy Cloning Kits are convenient to use containing pre cut dephosphorylated pJAZZ OC or pJAZZ OK cloning vector DNATerminator End Repair enzymes and buffer with the blunt kit only ligase and ligation buffer containing ATP sequencing primers competent cells and DNA controls Improvements over original BigEasy Kit The BigEasy v2 0 Kit contains two major changes from the original Big Easy Kit see Table below The new pJAZZ OC vector is resistant only to chloramphenicol and the new pJAZZ OK vector is resistant to only kanamycin The original pJAZZ KA vector was resistant to kanamycin plus ampicillin The new BigEasy TSATM Electrocompetent Cells have an ampicillin resistance gene integrated into the chromosome and no exogenous plasmids The original BigEasy pTel cells had an integrated chloramphenicol gene and an exogenous plasmid that encoded gentamycin resistance Kit Version Vector Cells BigEasy v2 0 pJAZZ OC chloramphenicol BigEasy TSA ampicillin pJAZZ OK kanamycin No Endogenous plasmids Original BigEasy pJAZZ KA BigEasy pTel chloramphenicol kanamycin ampicillin gentamycin on endogenous plasmid Lucigen Corporation 5 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits
47. ibiotic used Use chloramphenicol for pJAZZ OC vector use kanamycin for the pJAZZ OK vector Incorrect amounts of antibiotic in agar plates Add the correct amount of chloramphenicol or kanamycin to molten agar at 55 C before pouring plates see Appendix A DO NOT spread antibiotic onto the surface of agar plates High background of transformants that do not contain Contaminating enzymes in ligation reaction Purify DNA after DNA End Repair reaction DO NOT add T4 Polynucleotide Kinase to the ligation reaction inserts Contaminating oligo nucleotides in ligation reaction Use multiple methods of size selection e g column plus agarose gel For purification of fragments from agarose gels run gels without Ethidium Bromide followed by post staining Incorrect amount of antibiotic in agar plates DO NOT spread antibiotic onto the surface of agar plates Add the correct amount of chloramphenicol or kanamycin to molten agar at 55 C before pouring plates see Appendix A Lucigen Corporation 888 575 9695 608 831 9011 www lucigen com MA033 v2 2 BigEasy v2 0 Linear Cloning Kits Appendix F Sequence of pJAZZ OC vector 12887 bp lacZ stuffer fragment is underlined GCGTATAA GGAC ATTGTGTGC CTATTATG TT TCTC GGGGGAACCGGGA CAAGGCGACAAAACGAAGAGAA GCAGAAAAGAAT TA GATAAGGAG AGTGTGACGAAAGCAGCATAA AACAT AAGCGCAGAACAATA
48. ication The copy number is 2 4 cell prior to induction it is increased by approximately 5 20 fold by induction in BigEasy TSA cells see below The GenBank Accession number of the pJAZZ OC vector is EF583812 The Accession number for the pJAZZ OK vector will be available shortly The DNA sequences are also provided in Appendices F and G Lucigen Corporation 6 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits BigEasy TSA Electrocompetent Cells The BigEasy TSA strain is derived from Lucigen s E cloni 10G cells This strain contains several genes from phage N15 which are essential for high transformation efficiency and induction of copy number of the pJAZZ linear vectors Transformation with other strains will be 20 200 X less efficient The N15 genes are teN which encodes pro telomerase for efficient replication of the linear plasmid the sopAB genes for stable inheritance and the antA gene to regulate copy number These cells give high yield and high quality plasmid DNA due to the endA1 and recA1 mutations They contain the mcr and mrr mutations which allow stable cloning of methylated genomic DNA that has been isolated directly from mammalian or plant cells BigEasy TSA cells contain an integrated copy of the bla gene they are therefore resistant to ampicillin BigEasy TSA Genotype F mcrA A mrr hsdRMS mcrBC 80dlacZAM15 AlacX74 endA1 recA1araD139 A ara leu 7697 galU gak rpsL nupG
49. it Instruction Manual This limited license specifically excludes manufacture of pJAZZ vector or BigEasy competent cells or any derivatives thereof The buyer cannot modify the pJAZZ vector sequence s contained in this product for any purpose without express written consent of Lucigen Corp The buyer can not modify the BigEasy competent cell strain s contained in this product without the written consent of Lucigen Corp Lucigen Corporation reserves all other rights in particular the purchaser of this product may not transfer or otherwise sell this product or its components or derivatives to a third party and no rights are conveyed to the purchaser to use the product or its components or derivatives for commercial purposes The buyer may transfer information or materials made through the employment of this product or its components to a scientific collaborator provided that such transfer is not for commercial purposes and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for commercial purposes Commercial purposes includes any activity for which a party receives consideration and may include but is not limited to 1 use of the product or its components or derivatives in manufacturing 2 use of the product or its components or derivatives for diagnostic purposes 3 transfer or sale of vectors made with the product or
50. itions The Ligation Components of the BigEasy Linear Cloning Kits are shipped in Container 1 which should be stored at 20 C BigEasy TSA Electrocompetent Cells are shipped in Container 2 which must be stored at 80 C The DNATerminator Kit provided with the Blunt digest of the vector is shipped in Container 3 and should be stored at 20 C BigEasy TSA Electrocompetent Cells and DNATerminator Kits may be purchased separately Container 1 BigEasy Ligation Components Store at 20 C 5 Reactions 10 Reactions 20 Reactions pJAZZ OC or OK Vector Notl ends 50 ng ul or 5 ul 10 ul 2 x 10 ul pJAZZ OC or OK Vector Blunt ends 100 ng ul CloneSmart DNA Ligase 2 U ul 12 ul 12 ul 2 x12 ul CloneDirect 10X Ligation Buffer includes ATP 100 ul 100 ul 2 x 100 ul Positive Control Insert DNA 5 ul 5 ul 2x5y Includes one type of insert control lambda Pmel Smal blunt 500 ng ul Or lambda PspOMI Eagl Notl compatible 500 ng ul BigEasy Sequencing Primers 200 reactions each SL1 Primer 3 2 pmol ul 2 x 200 ul NZ RevC Primer 3 2 pmol l 2 x 200 ul Container 2 BigEasy TSA Electrocompetent Cells Store at 80 C BigEasy TSA Electrocompetent Cells SOLOs Store at 80 C Transformation Control pKanR DNA 1 ng ul Store at 20 C or 80 C Arabinose Induction Solution 1000 X Store at 20 C or 80 C Recovery Medium Store at 20 C or 80 C 96 8 x 12 ml YT Agar powder Lucig
51. lumn elution buffer e g Qiagen Buffer EB or 10 mM Tris pH8 5 2 Ligation to the pJAZZ Vector In the BigEasy ligation reaction the pre processed pJAZZ vector is ligated with phosphorylated insert fragments in a total volume of 10 ul For library construction we recommend using 200 500 ng of insert DNA in the size range of 1 40 kb For cloning a single DNA species 100 ng of insert is usually sufficient Successful cloning can be achieved routinely with less than 100 ng of insert but use of low amounts of insert will result in significantly fewer transformants The ligation is performed as follows 1 Before ligation run a sample of the insert DNA on an agarose gel to verify the quantity and integrity of the fragment 2 Briefly centrifuge the tube containing the pJAZZ vector Mix by gently pipeting up and down several times Likewise centrifuge and mix the CloneDirect Buffer 3 Combine the following components in a 1 5 ml tube adding the CloneSmart DNA Ligase last Notl Ligation Blunt Ligation Insert DNA 100 500 ng 5 phosphorylated Notl or Blunt ends X ul X ul pJAZZ OC or OK Vector 1 0 ul 1 0 ul 10X CloneDirect Ligation buffer contains ATP 1 0 ul 1 0 ul CloneSmart DNA Ligase 2 U ul 1 0 ul 1 0 ul H20 y ul y ul Total reaction volume 10 0 ul 10 0 ul For Blunt ligation up to 2 0 ul of vector may be used The number of clones will increase proportionately with the amount of vector The volume
52. of the ligation reaction may be scaled up if necessary 4 Mix by gently pipeting the reaction mixture up and down Incubate at room temperature 21 25 C for 2 hours Optional control reactions include the following Positive Control Insert DNA To determine the ligation and transformation efficiency with a known insert use 1 ul 500 ng of the supplied contro DNA Vector Background To determine the background of empty vector omit Insert DNA in the above reaction Lucigen Corporation 11 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Preparation for Transformation 1 Prepare YT Agar from powder included with cells Add appropriate antibiotic as per Appendix A 2 Essential Heat denature the ligation reaction at 70 C for 15 minutes 3 Cool to room temperature for 15 seconds followed by 0 4 C for 15 seconds to condense water vapor inside the tube 4 Spin 1 minute at 12 000 rpm to collect condensation and pellet precipitated material 5 The soluble sample is ready for transformation precipitating the DNA is not necessary 3 Transformation of BigEasy TSA Electrocompetent Cells Lucigen s BigEasy TSA Electrocompetent Cells must be used for high efficiency transformation with pJAZZ ligation reactions These cells yield gt 4 X 10 cfu ug of supercoiled control plasmid Electroporation BigEasy TSA Electrocompetent Cells are provided in 25 ul aliquots SOLOS su
53. pJAZZ OC and pJAZZ OK Vectors The pJAZZ OC and OK vectors are supplied pre digested at Smal blunt or Notl sites and they have dephosphorylated ends Each of the vectors contains a pair of nearly identical Multiple Cloning Sites on either side of the acZ reporter gene Figure 1 During preparation of the vector both Multiple Cloning Sites are cleaved by restriction digestion which completely removes the acZ marker gene and its promoter from the left and right vector arms The fragments are then dephosphorylated preventing their re ligation Insert DNA is ligated between the two arms to re create a viable linear plasmid Multiple A pJAZZ vectors before digestion 13 kb Cloning Site T T T _ __ ap LIEB Gam gy pJAZZ 0C lt Kant pJAZZ 0K B pJAZZ vectors after digestion 10 2 kb T T T Prep jeg insert OWA Gam DB pJAZZ 0C lt f s Kant pJAZZ 0K Figure 1 The pJAZZ vectors before digestion A and after digestion B During cloning the lacz fragment is replaced by the experimental insert DNA Tel protelomerase gene repA replication factor gene and origin of replication Cam chloramphenicol resistance gene Kan kanamycin resistance gene Approximate positions of transcriptional terminators are indicated After digestion the lacZ fragment is dephosphorylated to prevent its ligation into the vector but it is NOT removed from the preparation The pJAZZ OC and OK vectors also emplo
54. plus Notl Control Insert gt 500 000 gt 95 inserts No Insert Control Vector Background lt 5 000 lt 5 background pKanR Transformation Plasmid Control 10 pg NA gt 4 x 10 cfu ug plasmid 1 Results with experimental DNA may vary significantly particularly with larger insert sizes skewed base composition encoded peptides etc 2 A 50 ul aliquot of the empty vector control reaction should produce lt 25 colonies representing less than 5 background Lucigen Corporation 13 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits 3 A2 ul aliquot of transformed cells from the supercoiled control reaction diluted into 90 ul of TB should yield gt 800 colonies or gt 4 x 101 colonies per ug plasmid Use of too little insert DNA or insert DNA that is improperly end repaired or modified DNA that is not repairable yields significantly lower recombinant cloning efficiencies Cloning AT rich DNA and other recalcitrant sequences may also lead to fewer colonies With relatively few recombinant clones the number of empty vector colonies becomes noticeable in these cases For example if the Experimental Insert ligation reaction produces only 250 colonies from 50 ul of cells plated then the 25 colonies obtained from 50 ul of the No Insert Control ligation will represent a background of 10 See Appendix E for additional troubleshooting advice if necessary Screening The BigEasy sys
55. purified after digestion by standard methods e g agarose gel electrophoresis or binding to a purification column After purification proceed with Ligation to the pJAZZ Vector p 10 B Generation of Blunt Fragments Restriction Fragments DNA fragments created by digestion with blunt cutting restriction enzymes e g EcoRV or Hincll should be purified by standard methods e g agarose gel electrophoresis or binding to a purification column After purification proceed with Ligation to the pJAZZ Vector p 10 Restriction fragments that have 3 or 5 overhangs must undergo an end repair reaction to generate blunt ends prior to ligation Lucigen s DNATerminator Kit is supplied with the BigEasy Kit to repair these fragments see below Blunt PCR products PCR fragments created with proofreading polymerases such as Vent Phusion or Pfu polymerase have blunt ends However it is essential that the PCR products also have 5 phosphate groups The simplest method to generate phosphorylated PCR products is to perform the reaction with phosphorylated primers The primers can be synthesized directly with terminal 5 phosphate groups or they can be treated briefly with T4 PNK plus ATP prior to the PCR The following is a protocol for phosphorylation of primers Primer kinase reaction 4 ul Forward primer 100 pmol ul 4 ul Reverse primer 100 pmol ul 1 ul 10 X T4 PNK buffer mix containing ATP final conc 1 mM 1 ul
56. s Table of Contents Kit DESIGN AMONG ood atus dade HHHH E E HHHH HHHH 4 Components amp Storage Conditions kk kk kk keke kK kK KK KK 4 Kil DS BHO uie societe KANA A dM SN IM ME cute tec ets bas 5 pJAZZ Vectors l keke keke keka kk k kk kk kak kak kaka rk rk kra rk rak rak kk 6 BigEasy TSA Electrocompetent Cells 7 Purification and Size Fractionation of D NA kk kk kk kk KK nnn 7 Sensitivity of DNA to Short Wavelength UV Light 7 Materials and Equipment Needed a a 8 OVEIMIOW Ol Protoeol u n uu uu uska aka eka aka aka oba aka am 8 Biz T M PH 9 Preparation and Purification of Fragments for Cloning 9 End Repair of Fragments for Blunt Cloning k se 10 Ligation to the pJAZZ Vectors 11 Transformation of BigEasy TSA Electrocompetent Cells 12 SOreenilig u unn cet EA Rete tet AE te ie a raa a Yak wed an dana a za dane dan daman a Anf 14 DNA Isolation amp Sequencing oae ia oet eii Dato UI OM KK KK KK dins 14 Referen ES o TE 14 Appendix A Media Recipes
57. s The University of Oklahoma Health Sciences Center OKC www microgen ouhsc edu Lucigen Corporation 24 888 575 9695 608 831 9011 MA033 v2 2 www lucigen com
58. tem typically delivers gt 95 recombinant clones Insert DNAs that are large or have unusual base composition may produce very few colonies in which case screening by insert size may be necessary to detect the recombinant plasmids Digestion of mini preps with Notl will release the insert DNA from the vector arms See Figure 1B The Notl fragment from the left arm is 10 kb and from the right arm is 2 2 kb DNA Isolation amp Sequencing Grow transformants in TB medium plus 12 5 pg ml chloramphenicol The BigEasy TSA Electrocompetent Cells are recA endA deficient and will provide high quality plasmid DNA Standard alkaline lysis methods of plasmid preparation are effective for isolation of linear pJAZZ clones For most clones Induction Solution can be added to the culture medium before use Overnight induction will yield approximately 5 20 ug of linear plasmid DNA per 1 ml culture Without induction the pJAZZ vector yields 0 5 2 ug per ml of culture In either case yields generally decrease with larger inserts Approximately 150 400 ng of recombinant plasmid is sufficient for sequencing with the higher range of template required for larger inserts Standard protocols for cycle sequencing work well for the pJAZZ vector The BigEasy Kit is provided with the sequencing primers SL1 and NZ RevC The sequence of the primers and their orientation relative to the pJAZZ plasmid is shown in Appendix D References 1 Ravin NV Ravin VK 1999 Use of
59. tes at room temperature Stop the reaction by incubation at 70 C for 15 minutes Lucigen Corporation 10 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits IMPORTANT Do not exceed the recommended enzymatic treatment of the fragment Excessive enzyme treatment can lead to nucleolytic degradation of the fragments For less than 0 2 pmol of DNA the amount of enzyme may be scaled down and the time decreased to 15 minutes The heat denaturation step may be omitted if the reaction is stopped and the DNA is immediately purified by addition of a protein denaturing reagent e g phenol or guanidinium HCI Purification of Repaired Fragments If repaired or kinased fragments are subsequently fractionated by gel electrophoresis no further purification is necessary to remove the repair enzymes Use of short wavelength UV light e g 254 302 or 312 nm must be avoided After electrophoresis DNA may be isolated using your method of choice If the DNA is not fractionated by electrophoresis after end repair it must be purified by extraction or binding to a purification column to remove the repair enzymes Heat denaturation is NOT sufficient to inactivate the end repair enzymes Failure to completely remove residual enzymes may result in a large background of empty vector clones or greatly decreased ligation efficiency Elute or resuspend the DNA at a concentration of at least 30 ng ul in deionized water or co
60. vettes for electrocompetent cells Successful results are obtained with cuvettes from Eppendorf Cat 4307 000 569 BTX Model 610 or BioRad Cat 11165 2089 Users have reported difficulties using Lucigen s electrocompetent cells with Invitrogen cuvettes Cat 65 0030 e Sterile 17 x 100 mm culture tubes e Prepare YT Agar from powder included in the kit e YT CXI agar plates containing chloramphenicol plus XGAL IPTG See Appendix A e YT KXI agar plates containing kanamycin plus XGAL IPTG See Appendix A Note Colony growth may be slow or variable on LB agar plates Overview of the BigEasy v2 0 Cloning Process 1 Preparation and Purification of DNA Fragments for Cloning A Notl ends B Blunt ends Restriction fragments Blunt PCR products from proofreading PCR enzymes Tailed PCR products from non proofreading PCR enzymes Mechanically sheared fragments C End repair of fragments for blunt cloning 2 Ligation of DNA Fragments to pJAZZ vector 3 Transformation of BigEasy TSA Electrocompetent Cells Lucigen Corporation 8 888 575 9695 608 831 9011 MAO33 v2 2 www lucigen com BigEasy v2 0 Linear Cloning Kits Detailed Protocol 1 Preparation and Purification of DNA Fragments for Cloning A Generation of Notl Restriction Fragments DNA fragments created by digestion with Notl can be cloned directly into the pJAZZ OC Not or pJAZZ OK Notl vector preparation For optimum results the DNA should be
61. w England Biolabs Inc Phusion is a trademark of Finnzymes Oy Lucigen s products are sold for research use only and are not to be used in humans or for medical diagnostics Lucigen s liability with respect to any BigEasy product is limited to the replacement of the product No other warranties of any kind expressed or implied including without limitation any implied fitness for any particular use are provided by Lucigen Lucigen is not liable for any direct indirect incidental or consequential damages arising out of or in connection with the use or inability to use any of its BigEasy products Limited Label License This product is the subject of U S Patent 6 709 861 and pending patent applications owned by Lucigen Corporation The consideration paid for this product grants a Limited License to use the product pursuant to the terms set forth in this Limited Label License Academic Not for Profit and For Profit institutions acquire certain limited nontransferable rights with the purchase of this product see below The purchase of this product does not convey a license under any of the claims in the foregoing patent or patent applications By use of this product you accept the terms and conditions of the Limited Label License The purchase price of this product includes limited nontransferable rights to use only the purchased amount of the product to perform BigEasy Linear Cloning Technology and only as described in the BigEasy Linear Cloning K
62. ximately 20 25 ml per petri plate YT Agar per liter Mix 8 g Bacto tryptone 5 g yeast extract 5 g NaCl 15 g agar autoclave and cool to 55 C TB Culture Medium Per liter 11 8 g Bacto tryptone 23 6 g yeast extract 9 4 g dipotassium hydrogen phosphate Ko HPO anhydrous 2 2 g potassium dihydrogen phosphate KH2PQO anhydrous 0 4 glycerol Mix all components except glycerol autoclave and cool to 55 C Add 8 ml filter sterilized 5096 glycerol per liter prior to using Arabinose Induction Solution 1000X stock Dissolve L arabinose in water to 10 w v to make a 1000X stock Filter sterilize Growing Transformed Cultures Colonies obtained from a pJAZZ transformation can be further grown in TB or LB culture medium containing the 12 5 ug ml chloramphenicol Add 1 1000 volume of Induction Solution to the medium for increased copy number Transformed cultures can be stored by adding sterile glycerol to 20 final concentration and freezing at 70 C Unused portions of the ligation reactions may be stored indefinitely at 20 C Appendix B pSMART Application Guide Numerous cloning kits are available from Lucigen to accommodate any cloning situation For routine applications we recommend using the CloneSmart HCKan Blunt Cloning Kit containing the high copy number pSMART HCKan vector For cloning toxic genes or more difficult DNA sequences we recommend using the low copy vector in the CloneSmart LCKan Blunt Cloning Kit The pSM
63. y the CloneSmart transcription free cloning technology US Patent 6 709 861 which eliminates transcription both into and out of the insert DNA During preparation of the vector the acZ promoter is excised along with the coding region Thus cloned fragments are not subjected to vector driven transcription In conventional plasmids inserts are cloned downstream of a strong promoter within the coding sequence of acZ or a negative selection gene such as ccaB Transcription from the promoter causes loss of plasmids containing toxic coding sequences strong secondary structure or other deleterious features In the pJAZZ vectors as in most Lucigen vectors transcription across the insert is avoided so this loss is minimized Inserts containing E coli ike promoters are often difficult to clone in conventional plasmids because transcription from these promoters can interfere with the plasmid s replication or expression of its drug resistance gene In Lucigen s pJAZZ vectors strong transcription terminators flank the cloning site to block this transcription eliminating another source of cloning bias and sequencing gaps The left arm of the vector contains the origin of replication and the right arm encodes resistance to chloramphenicol or kanamycin Figure 1 Selection with the appropriate antibiotic results in recombinant clones containing both vector arms flanking the insert DNA The pJAZZ OC and OK vectors contain an inducible origin of repl

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