分子生物学实验专题手册.ai

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2 >> 2002 Piscataway CRO OptimumGene TM CloneEZ ONE-HOUR Western TM THE TM Epitope Tag

3 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

4 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

5 μl μl 2+ μl~ μl~~ r μlμg μl ~ μl μl μl μl μl μg μl μl ~ ~

6 2. PCR ~ ~ ~ Taq μl ~ ~μ μ 2+ ~ 2+ Taq ~~~Taq ~ Taq ~ ~ ~ ~ Taq ~

7 μgμg ~0.4 μg/μl

8 ~ ~

9 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

10 μ μ

11 μl μl μl μl μl μl

12 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

13 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

14 ' '

16 ''' ' 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 (80%~90%)

17 μl μlμl μgμmol μmolμmol μmpmol/μl

18 MW= A G C T M R W S Y K V H D B N Ns M=A/C=( )/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 '-TAMARA '-(6 FAM) '-DABCYL '-HEX '-(6 FAM) AAAAAAAAAAAAAAAAAAAA '-TET '-Amino Modifier C GGGGGGGGGGGGGGGGGGGG '-Cy '-Amino Modifier C CCCCCCCCCCCCCCCCCCCC '-Cy '-Thiol Modifier C TTTTTTTTTTTTTTTTTTTT-3 5-AAAAAGGGGGTTTTTCCCCC μmol/l μlμl 100 μmol/l pmol/μl

19 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

20 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

21 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'

22 μl μl ng/μl μl ng/μlμl pmol/μl μg μg pmol/μl μl ng/μlμl pmol/μl ng/μl μl pmol/μl

23 ng/μl 100 ng/μl μlng/μ μl μl μ

26 EditSeq: MapDraw: GeneQuest: MegAlign: Protean: PrimerSelect: SeqMan II:

..I II GST GST

..I II GST GST 10384 9826012 UDC 2001 6 2001 7 2001 7 ..I II..... 1 1.1...1 1.2 3 1.3. 11 1.4...12 13 2.1.13 2.2 17.23 3.1 23 3.2 23 3.3 24 3.4...27 30 3.6..31 3.7..32.34 4.1 GST-..34 4.2 GST-..35 4.3 36.. 39 5.1.. 39