Product Data Sheet

Standard heat-shock transformation of chemically competent bacteria

1. Take competent cells out of -80°C and thaw on ice.
2. Take agar plates (containing the appropriate antibiotic) out of 4°C to warm up to room temperature or place in 37°C incubator.
3. Mix 5μl of DNA into 100μL competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4. Place the competent cell/DNA mixture on ice for 20-30min.
5. Heat shock each transformation tube by placing the bottom 2/3 of the tube into a 42°C water bath for 42 seconds. Put the tubes back on ice for 2 min.
6. Add 250-500μl SOC media (without antibiotic) and grow in 37°C shaking incubator for 45min.
7. Plate all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
8. Incubate plates at 37°C overnight.

质粒描述：

pBeloBAC11 is an E. coli plasmid cloning vector designed for the construction of Bacterial Artificial Chromosomes (BACs). It is maintained in single copy, which allows the cloning and stable maintenance of very large DNA fragments (up to 300 kb; 1).Based on the Ori2 (OriS) replicon of the F (fertility) factor of E. coli, the vector encodes the SopAB functions for active partitioning (2). These functions act at SopC to ensure that each daughter cell gets a copy of the plasmid. Initiation factor RepE (also known as RepA) mediates assembly of a replication complex at Ori2 (3-5).The cloning region includes the following features: unique cloning sites BamHI, SphI and HindIII in a lacZα gene allowing for insert screening by α-complementation; T7 and SP6 phage promoters reading into the cloning sites for generation of RNA probes for blot procedures; several GC-rich restriction sites flanking the cloning segment for removal of the cloned insert; a chloramphenicol selectable marker; a lambda cos site for packaging into phage lambda particles if desired and also assuring a unique cleavage site for mapping; and a loxP site for specific cleavage by Cre recombinase in the presence of loxP oligonucleotide (6) Enzymes with unique restriction sites are shown in bold type. Location of sites of all NEB restriction enzymes can be found on the NEB web site (choose Technical Reference > DNA Sequences and Maps). Restriction site coordinates refer to the position of the 5´-most base on the top strand in each recognition sequence.Open reading frame (ORF) coordinates are in the form "translational start – translational stop”; numbers refer to positions on the top (clockwise) strand, regardless of the direction of transcription and include the startand stop codons.Lambda cos site coordinates are the boundaries of the HincII fragment surrounding the annealed 12 base overhangs.

There are no restriction sites for the following enzymes:

 AarI(x), AatII, AscI, AsiSI, AvrII, BbvCI, BciVI,

BmtI, BsiWI, BspDI, BstBI, Bsu36I, ClaI, FseI, I-CeuI, I-SceI,

MluI, NheI, NsiI, PI-PspI, PI-SceI, PacI, PmeI, PmlI, RsrII,

SacII, SanDI(x), SwaI, XcmI, ZraI.

(x) = enzyme not available from NEB

>sequence  7507bps

1 gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
       61 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
      121 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
      181 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
      241 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
      301 ccagtgaatt gtaatacgac tcactatagg gcgaattcga gctcggtacc cggggatcct
      361 ctagagtcga cctgcaggca tgcaagcttg agtattctat agtgtcacct aaatagcttg
      421 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      481 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      541 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      601 cattaatgaa tcggccaacg cgaacccctt gcggccgccc gggccgtcga ccaattctca
      661 tgtttgacag cttatcatcg aatttctgcc attcatccgc ttattatcac ttattcaggc
      721 gtagcaacca ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg
      781 ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
      841 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
      901 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
      961 actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt
     1021 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     1081 ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg
     1141 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     1201 catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     1261 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     1321 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     1381 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     1441 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     1501 aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg
     1561 cttcccggta tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac
     1621 aggtatttat tcgcgataag ctcatggagc ggcgtaaccg tcgcacagga aggacagaga
     1681 aagcgcggat ctgggaagtg acggacagaa cggtcaggac ctggattggg gaggcggttg
     1741 ccgccgctgc tgctgacggt gtgacgttct ctgttccggt cacaccacat acgttccgcc
     1801 attcctatgc gatgcacatg ctgtatgccg gtataccgct gaaagttctg caaagcctga
     1861 tgggacataa gtccatcagt tcaacggaag tctacacgaa ggtttttgcg ctggatgtgg
     1921 ctgcccggca ccgggtgcag tttgcgatgc cggagtctga tgcggttgcg atgctgaaac
     1981 aattatcctg agaataaatg ccttggcctt tatatggaaa tgtggaactg agtggatatg
     2041 ctgtttttgt ctgttaaaca gagaagctgg ctgttatcca ctgagaagcg aacgaaacag
     2101 tcgggaaaat ctcccattat cgtagagatc cgcattatta atctcaggag cctgtgtagc
     2161 gtttatagga agtagtgttc tgtcatgatg cctgcaagcg gtaacgaaaa cgatttgaat
     2221 atgccttcag gaacaataga aatcttcgtg cggtgttacg ttgaagtgga gcggattatg
     2281 tcagcaatgg acagaacaac ctaatgaaca cagaaccatg atgtggtctg tccttttaca
     2341 gccagtagtg ctcgccgcag tcgagcgaca gggcgaagcc ctcgagtgag cgaggaagca
     2401 ccagggaaca gcacttatat attctgctta cacacgatgc ctgaaaaaac ttcccttggg
     2461 gttatccact tatccacggg gatattttta taattatttt ttttatagtt tttagatctt
     2521 cttttttaga gcgccttgta ggcctttatc catgctggtt ctagagaagg tgttgtgaca
     2581 aattgccctt tcagtgtgac aaatcaccct caaatgacag tcctgtctgt gacaaattgc
     2641 ccttaaccct gtgacaaatt gccctcagaa gaagctgttt tttcacaaag ttatccctgc
     2701 ttattgactc ttttttattt agtgtgacaa tctaaaaact tgtcacactt cacatggatc
     2761 tgtcatggcg gaaacagcgg ttatcaatca caagaaacgt aaaaatagcc cgcgaatcgt
     2821 ccagtcaaac gacctcactg aggcggcata tagtctctcc cgggatcaaa aacgtatgct
     2881 gtatctgttc gttgaccaga tcagaaaatc tgatggcacc ctacaggaac atgacggtat
     2941 ctgcgagatc catgttgcta aatatgctga aatattcgga ttgacctctg cggaagccag
     3001 taaggatata cggcaggcat tgaagagttt cgcggggaag gaagtggttt tttatcgccc
     3061 tgaagaggat gccggcgatg aaaaaggcta tgaatctttt ccttggttta tcaaacgtgc
     3121 gcacagtcca tccagagggc tttacagtgt acatatcaac ccatatctca ttcccttctt
     3181 tatcgggtta cagaaccggt ttacgcagtt tcggcttagt gaaacaaaag aaatcaccaa
     3241 tccgtatgcc atgcgtttat acgaatccct gtgtcagtat cgtaagccgg atggctcagg
     3301 catcgtctct ctgaaaatcg actggatcat agagcgttac cagctgcctc aaagttacca
     3361 gcgtatgcct gacttccgcc gccgcttcct gcaggtctgt gttaatgaga tcaacagcag
     3421 aactccaatg cgcctctcat acattgagaa aaagaaaggc cgccagacga ctcatatcgt
     3481 attttccttc cgcgatatca cttccatgac gacaggatag tctgagggtt atctgtcaca
     3541 gatttgaggg tggttcgtca catttgttct gacctactga gggtaatttg tcacagtttt
     3601 gctgtttcct tcagcctgca tggattttct catacttttt gaactgtaat ttttaaggaa
     3661 gccaaatttg agggcagttt gtcacagttg atttccttct ctttcccttc gtcatgtgac
     3721 ctgatatcgg gggttagttc gtcatcattg atgagggttg attatcacag tttattactc
     3781 tgaattggct atccgcgtgt gtacctctac ctggagtttt tcccacggtg gatatttctt
     3841 cttgcgctga gcgtaagagc tatctgacag aacagttctt ctttgcttcc tcgccagttc
     3901 gctcgctatg ctcggttaca cggctgcggc gagcgctagt gataataagt gactgaggta
     3961 tgtgctcttc ttatctcctt ttgtagtgtt gctcttattt taaacaactt tgcggttttt
     4021 tgatgacttt gcgattttgt tgttgctttg cagtaaattg caagatttaa taaaaaaacg
     4081 caaagcaatg attaaaggat gttcagaatg aaactcatgg aaacacttaa ccagtgcata
     4141 aacgctggtc atgaaatgac gaaggctatc gccattgcac agtttaatga tgacagcccg
     4201 gaagcgagga aaataacccg gcgctggaga ataggtgaag cagcggattt agttggggtt
     4261 tcttctcagg ctatcagaga tgccgagaaa gcagggcgac taccgcaccc ggatatggaa
     4321 attcgaggac gggttgagca acgtgttggt tatacaattg aacaaattaa tcatatgcgt
     4381 gatgtgtttg gtacgcgatt gcgacgtgct gaagacgtat ttccaccggt gatcggggtt
     4441 gctgcccata aaggtggcgt ttacaaaacc tcagtttctg ttcatcttgc tcaggatctg
     4501 gctctgaagg ggctacgtgt tttgctcgtg gaaggtaacg acccccaggg aacagcctca
     4561 atgtatcacg gatgggtacc agatcttcat attcatgcag aagacactct cctgcctttc
     4621 tatcttgggg aaaaggacga tgtcacttat gcaataaagc ccacttgctg gccggggctt
     4681 gacattattc cttcctgtct ggctctgcac cgtattgaaa ctgagttaat gggcaaattt
     4741 gatgaaggta aactgcccac cgatccacac ctgatgctcc gactggccat tgaaactgtt
     4801 gctcatgact atgatgtcat agttattgac agcgcgccta acctgggtat cggcacgatt
     4861 aatgtcgtat gtgctgctga tgtgctgatt gttcccacgc ctgctgagtt gtttgactac
     4921 acctccgcac tgcagttttt cgatatgctt cgtgatctgc tcaagaacgt tgatcttaaa
     4981 gggttcgagc ctgatgtacg tattttgctt accaaataca gcaatagtaa tggctctcag
     5041 tccccgtgga tggaggagca aattcgggat gcctggggaa gcatggttct aaaaaatgtt
     5101 gtacgtgaaa cggatgaagt tggtaaaggt cagatccgga tgagaactgt ttttgaacag
     5161 gccattgatc aacgctcttc aactggtgcc tggagaaatg ctctttctat ttgggaacct
     5221 gtctgcaatg aaattttcga tcgtctgatt aaaccacgct gggagattag ataatgaagc
     5281 gtgcgcctgt tattccaaaa catacgctca atactcaacc ggttgaagat acttcgttat
     5341 cgacaccagc tgccccgatg gtggattcgt taattgcgcg cgtaggagta atggctcgcg
     5401 gtaatgccat tactttgcct gtatgtggtc gggatgtgaa gtttactctt gaagtgctcc
     5461 ggggtgatag tgttgagaag acctctcggg tatggtcagg taatgaacgt gaccaggagc
     5521 tgcttactga ggacgcactg gatgatctca tcccttcttt tctactgact ggtcaacaga
     5581 caccggcgtt cggtcgaaga gtatctggtg tcatagaaat tgccgatggg agtcgccgtc
     5641 gtaaagctgc tgcacttacc gaaagtgatt atcgtgttct ggttggcgag ctggatgatg
     5701 agcagatggc tgcattatcc agattgggta acgattatcg cccaacaagt gcttatgaac
     5761 gtggtcagcg ttatgcaagc cgattgcaga atgaatttgc tggaaatatt tctgcgctgg
     5821 ctgatgcgga aaatatttca cgtaagatta ttacccgctg tatcaacacc gccaaattgc
     5881 ctaaatcagt tgttgctctt ttttctcacc ccggtgaact atctgcccgg tcaggtgatg
     5941 cacttcaaaa agcctttaca gataaagagg aattacttaa gcagcaggca tctaaccttc
     6001 atgagcagaa aaaagctggg gtgatatttg aagctgaaga agttatcact cttttaactt
     6061 ctgtgcttaa aacgtcatct gcatcaagaa ctagtttaag ctcacgacat cagtttgctc
     6121 ctggagcgac agtattgtat aagggcgata aaatggtgct taacctggac aggtctcgtg
     6181 ttccaactga gtgtatagag aaaattgagg ccattcttaa ggaacttgaa aagccagcac
     6241 cctgatgcga ccacgtttta gtctacgttt atctgtcttt acttaatgtc ctttgttaca
     6301 ggccagaaag cataactggc ctgaatattc tctctgggcc cactgttcca cttgtatcgt
     6361 cggtctgata atcagactgg gaccacggtc ccactcgtat cgtcggtctg attattagtc
     6421 tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac
     6481 tcgtatcgtc ggtctgataa tcagactggg accacggtcc cactcgtatc gtcggtctga
     6541 ttattagtct gggaccatgg tcccactcgt atcgtcggtc tgattattag tctgggacca
     6601 cggtcccact cgtatcgtcg gtctgattat tagtctggaa ccacggtccc actcgtatcg
     6661 tcggtctgat tattagtctg ggaccacggt cccactcgta tcgtcggtct gattattagt