>> 2002 Piscataway CRO OptimumGene TM CloneEZ ONE-HOUR Western TM THE TM Epitope Tag 36 36 39 40 41 41 41 45........................ 47 47 47 47 48............... 19 19 20 21 22............... 23 23 24 27 29 29 29 30 32 33 35................................. 2 2 2 2 3 3 5 5 7 7 8 13 13 13 14 17................................................
商品化载体 连 接 转化重组质粒 PCR 目的基因获取 PCR Taq 酶切载体及目的基因 细胞增殖 EcoRI 载体制备 5' G AATTC 3' 3' C TT AAG 5' EcoRI 5 μl+5 μl DNA Loading buffer20~30 min 1 TAE+ 0.1 μl/ml 45 μl ddh 2 O 25~30 μl ddh 2 O - 1 - - 2 -
20~30 μl ddh 2 O ~~ ~ 600 ~ ~ E. coli E. coli 10~100 2~10 μg/ml 100~200 μg/ml ~~ ~ μl μl μl μl ~ μl ~ μl μl μl - 3 - - 4 -
μl μl 2+ μl~ μl~~ r μlμg μl ~ μl μl μl μl μl μg μl μl ~ ~ - 5 - - 6 -
2. PCR ~ ~ ~ Taq μl ~ ~μ 2+ 2+ 2+ 2+ μ 2+ ~ 2+ Taq ~~~Taq ~ Taq ~ ~ ~ ~ Taq ~ 2+ - 7 - - 8 -
μgμg 2+ 2+ 0.25~0.4 μg/μl - 9 - - 10 -
~ ~ - 11 - - 12 -
μg/μl 5'...A C C G G T...3' 3'...T G G C C A...5' 5'...A A G C T T...3' 3'...T T C G A A...5' 5'...G C T A G C...3' 3'...C G A T C G...5' 5'...G T C G A C...3' 3'...C A G C T G...5' 5'...G G A T C C...3' 3'...C C T A G G...5' 5'...G T T A A C...3' 3'...C A A T T G...5' 5'...G C G G C C G C...3' 3'...C G C C G G C G...5' 5'...C C C G G G...3' 3'...G G G C C C...5' 5'...A G A T C T...3' 3'...T C T A G A...5' 5'...G G T A C C...3' 3'...C C A T G G...5' 5'...C T G C A G...3' 3'...G A C G T C...5' 5'...T C T A G A...3' 3'...A G A T C T...5' 5'...G A A T T C...3' 3'...C T T A A G...5' 5'...A C G C G T...3' 3'...T G C G C A...5' 5'...G A G C T C...3' 3'...C T C G A G...5' 5'...C T C G A G...3' 3'...G A G C T C...5' 5'...G A T A T C...3' 3'...C T A T A G...5' 5'...C C A T G G...3' 3'...G G T A C C...5' 5'...C C G C G G...3' 3'...G G C G C C...5' μg - 13 - - 14 -
μ μ - 15 - - 16 -
- 17 - - 18 - μl μl μl μl μl μl
E.coli DNA Transcription Translation Co-translational folding Transcription: Antiviral motifs Chi-site Polymerase slippage site... mrna Ribosome Protein folding: Codon context RNA secondary structure... Deviant folding Natural folding Nucleotide Translation: Codon usage bias mrna secondary structure GC content... - 19 - - 20 -
任意长度的基因和各种复杂基因 长达 50,000 bp 高 GC 含量 高重复序列 OptimumGene TM 密码子优化 从头开始基因合成 Gene-on-Demand CloneEZ 无缝 克隆 μg - 21 - - 22 -
- 23 - - 24 - ' '
- 25 - - 26 -
''' ' RPC PAGE HPLC <11 mer 11~40 mer 41~59 mer 60~110 mer PCR RPC ( 60mer) PAGE DNAPCR 50 mer 50 mer (<11 mer) HPLCPAGE - 28 - (80%~90%) - 27 -
μl μlμl μgμmol μmolμmol μmpmol/μl - 29 - - 30 -
MW= A 313.21 + G 329.21 + C 289.18 + T 304.2 + M 301.2 +R 321.21 + W 308.71 + S 309.2 +Y 296.69 + K 316.71 + V 310.53 + H 302.2 + D 315.54 + B 307.53 + N 308.95 +16 Ns + - 61.96 16 M=A/C=(313.21+289.18)/2M=A/C; R=A/G; W=A/T; S=G/C; Y=C/T; K=G/T; V=A/G/C; H=A/C/T; D=A/G/T; B=G/C/T; N=A/G/C/T. 5'-Biotin 447.11 3'-TAMARA 623.60 5'-(6 FAM) 537.46 3'-DABCYL 462.44 5'-HEX 744.13 3'-(6 FAM) 569.46 5-AAAAAAAAAAAAAAAAAAAA-3 2.50 5'-TET 675.24 3'-Amino Modifier C3 153.07 5-GGGGGGGGGGGGGGGGGGGG-3 1.85 5'-Cy5 644.70 3'-Amino Modifier C7 211.18 5-CCCCCCCCCCCCCCCCCCCC-3 1.15 5'-Cy3 618.70 3'-Thiol Modifier C3 154.12 5-TTTTTTTTTTTTTTTTTTTT-3 5-AAAAAGGGGGTTTTTCCCCC-3 1.14 1.66 μmol/l μlμl 100 μmol/l pmol/μl - 31 - - 32 -
pmol/μl pmol/μl pmol/μl 6-FAM 6-carboxy-fluorescein 494 nm 518 nm Green μl TET HEX TAMRA 5-tetrachloro-fluorescein 5-hexachloro-fluorescein tetramethyl-6-carboxyrhodamine 521 nm 535 nm 560 nm 538 nm 553 nm 582 nm Orange Pink Rose ROX 6-carboxy-x-rhodamine 587 nm 607 nm Red Cy3 Indodicarbocyanine 552 nm 570 nm Red Cy5 Indodicarbocyanine 643 nm 667 nm Violet Taq - 33 - - 34 -
pmol/μl μl M13F(-47)/M13F/T7 pbluescriptsk(+) pcdna3 pcdna3.1(+) pcdna3.0(+)/myc-his C/A/B pcdna3.1(+)/myc-his C/A/B/ LacZ pcdna3.1/his C/A/B/LacZ pcdna3.1/hygro(-)/(+)/lacz pcdna3.1/v5-his C/A/B/LacZ H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 H1.3F/pCMV-F/pEGFP-N-5/T7 M13R(-48)/M13R/T3 BGH pcdna3.1r/bgh BGH pcdna3.1r/bgh pcdna3.1r/bgh pcdna3.1r/bgh pcdna3.1r/bgh pcdna3.1/v5-his-topo/lacz H1.3F/pCMV-F/pEGFP-N-5/T7 pcdna3.1r/bgh pcdna3.1/zeo(-)/(+)/cat H1.3F/pCMV-F/pEGFP-N-5/T7 pcdna3.1r/bgh pcolddna- I/II/III/ IV pdream2.1 pcold-f H1.3F/pCMV-F/pEGFP-N-5/T7 pcold-r SP6 pegfp-n1,2,3 H1.3F/pCMV-F/pEGFP-N-5 pegfp-n-3 /pegfp-c-5 /pegfp-c-3' pet15b T7 T7ter pet20b T7 T7ter pet22b T7 T7ter pet28a T7 T7ter - 35 - - 36 -
pet28b T7 T7ter 5'AD 5'- AGG GAT GTT TAA TAC CAC TAC -3' M13R(-48) 5'- AGC GGA TAA CAA TTT CAC ACA GGA -3' pet30b T7 T7ter 3'AD 5'- AGA TGG TGC ACG ATG CAC AG -3' GLP1 5'- TGT ATC TTA TGG TAC TGT AAC TG -3' pfastbac Dual pegfp-c-3' PFast BAC-F 5AOX1 5'- GAC TGG TTC CAA TTG ACA AGC -3' GLP2 5'- CTT TAT GTT TTT GGC GTC TTC CA -3' pfastbac HT A,B,C pegfp-c-3' PFast BAC-F 3AOX1 5'- GCA AAT GGC ATT CTG ACA TCC -3' pbad Forward 5'- ATG CCA TAG CAT TTT TAT CC -3' pfastbac1 pegfp-c-3' PFast BAC-F A-FACTOR 5 -TAC TAT TGC CAG CAT TGC TGC -3' pbad Reverse 5'- GAT TTA ATC TGT ATC AGG -3' pgem-t Easy M13F(-47)/M13F/T7 M13R(-48)/M13R 3'BD 5'- TAA GAG TCA CTT TAA AAT TTG TAT C -3' pegfp-n-5' 5'- TGG GAG GTC TAT ATA AGC AGA G -3' pgex-4t-1/3 p-gex5 p-gex3 CMV-Forward 5'- CGC AAA TGG GCG GTA GGC GTG -3' pegfp-n-3' 5'- CGT CGC CGT CCA GCT CGA CCA G -3' pgs-21a p-gex5 T7ter CMV-R 5'- GTT CAC GGT GCC CTC C -3' pegfp-c-5' 5'- CAT GGT CCT GCT GGA GTT CGT G -3' pmd18-t M13F(-47)/M13F M13R(-48)/M13R PGEX-5 5'- GGG CTG GCA AGC CAC GTT TGG TG -3' pegfp-c-3' 5'- TAT GGC TGA TTA TGA TCA GT -3' pmd19-t M13F(-47)/M13F M13R(-48)/M13R PGEX-3' 5'- CCG GGA GCT GCA TGT GTC AGA GG -3' pfastbacf 5'- GGA TTA TTC ATA CCG TCC CA -3' ppcr script M13F(-47)/M13F/T7 M13R(-48)/M13R/T3 T3 5'- ATT AAC CCT CAC TAA AGG GA-3' pfastbacr 5'- CAA ATG TGG TAT GGC TGA TT -3' ppic3.5 5 AOX 3 AOX T7 5'- TAA TAC GAC TCA CTA TAG GG -3' RVP3 5'- CTA GCA AAA TAG GCT GTC CC -3' ppic9 5 AOX 3 AOX T7-Ter 5'- GCT AGT TAT TGC TCA GCG G -3' RVP4 5'- GAC GAT AGT CAT GCC CCG CG -3' ppicz C/A/B 5 AOX 3 AOX BGH 5'- TAG AAG GCA CAG TCG AGG -3' PQE30F 5 - TGA GCG GAT AAC AAT TTC AC-3' ppiczalpha C/A/B 5 AOX/α-Factor 3 AOX SP6 5'- GAT TTA GGT GAC ACT ATA G -3' PQE30R 5 - GTT CTG AGG TCA TTA CTG G -3' puc57 puc18 puc19 M13F(-47)/M13F M13F(-47)/M13F M13F(-47)/M13F M13R(-48)/M13R M13R(-48)/M13R M13R(-48)/M13R M13F M13R M13F(-47) 5'- TGT AAA ACG ACG GCC AGT -3' 5'- CAG GAA ACA GCT ATG AC -3' 5'- CGC CAG GGT TTT CCC AGT CAC GAC -3' Pcold-F Pcold-R S.TAG 5 - CAGTGTAGTAAGGCAAGTCC -3' 5 - GGGCCTGCGCATTCTCATTG -3' 5'- CGAACGCCAGCACATGGACA -3' 其它 ITS4 5'- TCCTCCGCTTATTGATATGC -3' H1.3F 5 - GACGTCAATGGGAGTTTGTT -3' 27F 5'- AGAGTTTGATCCTGGCTCAG -3' 1492R 5 - GGTTACCTTGTTACGACTT -3' ITS1 5'- TCCGTAGGTGAACCTGCGG -3' - 37 - - 38 -
μl μl ng/μl μl ng/μlμl pmol/μl μg μg pmol/μl μl ng/μlμl pmol/μl ng/μl μl pmol/μl - 39 - - 40 -
ng/μl 100 ng/μl μlng/μ μl μl μ - 41 - - 42 -
- 43 - - 44 -
- 45 - - 46 -
EditSeq: MapDraw: GeneQuest: MegAlign: Protean: PrimerSelect: SeqMan II: - 47 - - 48 -